WO1999051639A1 - An integrin heterodimer and a subunit thereof - Google Patents

An integrin heterodimer and a subunit thereof Download PDF

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Publication number
WO1999051639A1
WO1999051639A1 PCT/SE1999/000544 SE9900544W WO9951639A1 WO 1999051639 A1 WO1999051639 A1 WO 1999051639A1 SE 9900544 W SE9900544 W SE 9900544W WO 9951639 A1 WO9951639 A1 WO 9951639A1
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WIPO (PCT)
Prior art keywords
subunit
integrin
amino acid
seq
fragment
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Application number
PCT/SE1999/000544
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French (fr)
Inventor
Evy LUNDGREN-ÅKERLUND
Original Assignee
Cartela Ab
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Publication date
Priority claimed from SE9801164A external-priority patent/SE9801164D0/en
Priority claimed from SE9900319A external-priority patent/SE9900319D0/en
Priority to ES99919725T priority Critical patent/ES2396693T3/en
Priority to EP99919725A priority patent/EP1068238B1/en
Priority to JP2000542360A priority patent/JP4912528B2/en
Priority to CA2324381A priority patent/CA2324381C/en
Application filed by Cartela Ab filed Critical Cartela Ab
Priority to AU37380/99A priority patent/AU761430B2/en
Priority to US09/647,544 priority patent/US7029858B1/en
Priority to DK99919725.4T priority patent/DK1068238T3/en
Publication of WO1999051639A1 publication Critical patent/WO1999051639A1/en
Priority to US11/347,179 priority patent/US8048991B2/en
Priority to US13/226,323 priority patent/US20120136141A1/en
Priority to US13/651,602 priority patent/US8895253B2/en
Priority to US14/552,308 priority patent/US10081666B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the fragment is a peptide chosen from the group comprising peptides of the cytoplasmic domain, the I-domain and the spliced domain.
  • the frag- ment is a peptide comprising the amino acid sequence KLGFFAHKKIPEEEKREEKLEQ, or a fragment comprising the amino acid sequence from about amino acid No. 952 to 7 about amino acid No. 986 of SEQ ID No. 1, or a fragment comprising the amino acid sequence from about amino acid no. 140 to about amino acid no. 337 of SEQ ID no. 1.
  • the invention is also related to the use of an integrin subunit ⁇ lO or an integrin heterodimer comprising said subunit ⁇ lO and a subunit ⁇ as a target for anti- 11 adhesive drugs or molecules in tendon, ligament, skeletal muscle or other tissues where adhesion impairs the function of the tissue.
  • a still further aspect of the inventions relates to a method of using DNA or RNA encoding an integrin subunit ⁇ lO or homologues or fragments thereof as a molecular target.
  • a polynucleotide or oligonucleotide hybridises to the DNA or RNA encoding an integrin subunit ⁇ lO or homologues or fragments there ⁇ of, whereby said polynucleotide or oligonucleotide fails to hybridise to a DNA or RNA encoding en integrin subunit ⁇ l.
  • Bovine chondrocytes, human chondrocytes or human chondrosarcoma cells were isolated as described earlier [Holmvall et al, Exp Cell Res, 221, 496-503 (1995), Camper et al, JBC, 273, 20383-20389 (1998)].
  • a Triton X-100 lysate of bovine chondrocytes was applied to a fibronectin-Sepharose precolumn followed by a collagen 18 type Il-Sepharose column and the integrin subunit ⁇ lO was eluted from the collagen type II-column by EDTA (Camper et al, JBC, 273, 20383-20389 (1998) .

Abstract

A recombinant or isolated integrin heterodimer comprising a novel subunit α10 in association with a subunit β is described. The α10 integrin may be purified from bovine chondrocytes on a collagen-type-II affinity column. The integrin or the subunit α10 can be used as marker or target of all types of cells, e.g. of chondrocytes, osteoblasts and fibroblasts. The integrin or subunit α10 thereof can be used as marker or target in different physiological or therapeutic methods. They can also be used as active ingredients in pharmaceutical compositions and vaccines.

Description

AN INTEGRIN HETERODIMER AND A SUBUNIT THEREOF
FIELD OF THE INVENTION
The present invention relates to a recombinant or isolated integrin heterodimer comprising a subunit αlO and a subunit β, the subunit αlO thereof, homologues and fragments of said integrin and of said subunit αlO having similar biological activity, processes of producing the same, polynucleotides and oligonucleotides encoding the same, vectors and cells comprising the same, binding entities binding specifically to the same, and the use of the same.
BACKGROUND OF THE INVENTION
The integrins are a large family of transmembrane glycoproteins that mediate cell-cell and cell-matrix interactions (1-5) . All known members of this superfamily are non-covalently associated heterodimers composed of an α- and a β-subunit. At present, 8 β-subunits (βl-β8) (6) and 16 α-subunits ( l-α9, αv, αM, αL, αX, ctllb, αE and D) have been characterized (6-21), and these subunits associate to generate more than 20 different integrins. The βl-subunit has been shown to associate with ten different α-subunits, αl-α9 and αv, and to mediate interactions with extracellular matrix proteins such as colla- gens, laminins and fibronectin. The major collagen binding integrins are αlβl and α2βl (22-25) . The integrins α3βl and α9βl have also been reported to interact with collagen (26,27) although this interaction is not well understood (28). The extracellular N-terminal regions of the α and β integrin subunits are important in the binding of ligands (29,30). The N-terminal region of the α-subunits is composed of a seven-fold repeated sequence (12,31) containing FG and GAP consensus sequences. The repeats are predicted to fold into a β-propeller domain (32) with the last three or four repeats containing putative divalent cation binding sites. The α-integrin subunits αl, α2, αD, αE, αL, αM and αX contain a -200 amino acid inserted domain, the I-domain (A-domain) , which shows similarity to sequences in von Willebrand factor, cartilage matrix protein and complement factors C2 and B (33,34). The I-domain is localized between the second and third FG-GAP repeats, it contains a metal .on-dependent adhesion s_ite (MIDAS) and it is involved in binding of ligands (35-38) .
Chondrocytes, the only type of cells in cartilage, express a number of different integrins including αlβl, α2βl, α3βl, α5βl, α6βl, αvβ3, and αvβ5 (39-41) . It has been shown that αlβl and α2βl mediate chondrocyte inter- actions with collagen type II (25) which is one of the major components in cartilage. It has alsb been shown that α2βl is a receptor for the cartilage matrix protein chondroadherin (42).
SUMMARY OF THE INVENTION
The present invention relates to a novel collagen type II binding integrin, comprising a subunit αlO in association with a subunit β, especially subunit βl, but also other β-subunits may be contemplated. In preferred embodiments, this integrin has been isolated from human or bovine articular chondrocytes, and human chondrosar- coma cells.
The invention also encompasses integrin homologues of said integrin, isolated from other species, such as bovine integrin heterodimer comprising a subunit αlO in association with a subunit β, preferably βl, as well as homologues isolated from other types of human cells or from cells originating from other species.
The present invention relates in particular to a recombinant or isolated integrin subunit αlO comprising the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, and homologues and or fragments thereof having the 3 same biological activity.
The invention further relates to a process of producing a recombinant integrin subunit αlO comprising the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, or homologues or fragments thereof having similar biological activity, which process comprises the steps of a) isolating a polynucleotide comprising a nucleo- tide sequence coding for a integrin subunit αlO, or homo¬ logues or fragments thereof having similar biological activity, b) constructing an expression vector comprising the isolated polynucleotide, c) transforming a host cell with said expression vector, d) culturing said transformed host cell in a culture medium under conditions suitable for expression of integrin subunit αlO, or homologues or fragments thereof having similar biological activity, in said transformed host cell, and, optionally, e) isolating the integrin subunit αlO, or homologues or fragments thereof having the same biological activity, from said transformed host cell or said culture medium.
The integrin subunit αlO, or homologues or fragments thereof having the same biological activity, can also be provided by isolation from a cell in which they are naturally present.
The invention also relates to an isolated polynucleotide comprising a nucleotide coding for a integrin subunit αlO, or homologues or fragments thereof having similar biological activity, which polynucleotide comprises the nucleotide sequence shown in SEQ ID No. 1 or SEQ ID No. 2, or parts thereof.
The invention further relates to an isolated polynucleotide or oligonucleotide which hybridises to a DNA or RNA encoding an integrin subunit αlO, having the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, or homologues or fragments thereof, wherein said polyoligo- 4 nucleotide or oligonucleotide fails to hybridise to a DNA or RNA encoding the integrin subunit αl .
The invention relates in a further aspect to vectors comprising the above polynucleotides, and to cells con- taining said vectors and cells that have polynucleotides or oligonycleotides as shown in SEQ ID No. 1 or 2 integrated in their genome.
The invention also relates to binding entities having the capability of binding specifically to the inte- grin subunit αlO or to homologues or fragments thereof, such as proteins, peptides, carbohydrates, lipids, natural ligands, polyclonal antibodies or monoclonal antibodies.
In a further aspect, the invention relates to a recombinant or isolated integrin heterodimer comprising a subunit αlO and a subunit β, in which the subunit αlO comprises the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, or homologues or fragments thereof having similar biological activity. In a preferred embodiment thereof, the subunit β is βl.
The invention also relates to a process of producing a recombinant integrin heterodimer comprising a subunit αlO and a subunit β, in which the subunit αlO comprises the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, which process comprises the steps of a) isolating one polynucleotide comprising a nucleotide sequence coding for a subunit αlO of an integrin heterodimer and, optionally, another polynucleotide com- prising a nucleotide sequence coding for a subunit β of an integrin heterodimer, or for homologues or fragments thereof having similar biological activity, b) constructing an expression vector comprising said isolated polynucleotide coding for said subunit αlO in combination with an expression vector comprising said isolated nucleotide coding for said subunit β, 5 c) transforming a host cell with said expression vectors, d) culturing said transformed host cell in a culture medium under conditions suitable for expression of an integrin heterodimer comprising a subunit αlO and a subunit β, or homologues or fragments thereof similar biological activity, in said transformed host cell, and, optionally, e) isolating the integrin heterodimer comprising a subunit αlO and a subunit β, or homologues or fragments thereof having the same biological activity, from said transformed host cell or said culture medium.
The integrin heterodimer, or homologues or fragments thereof having similar biological activity, can also be provided by isolation from a cell in which they are naturally present.
The invention further relates to a cell containing a first vector, said first vector comprising a polynucleotide coding for a subunit αlO of an integrin heterodimer, or for homologues or parts thereof having similar biological activity, which polynucleotide comprises the nucleotide sequence shown in SEQ ID No. 1 or SEQ ID No. 2 or parts thereof, and, optionally, a second vector, said second vector comprising a polynucleotide coding for a subunit β of an integrin heterodimer, or for homologues or fragments thereof.
In still another aspect, the invention relates to binding entities having the capability of binding specifically to the integrin heterodimer comprising a subunit αlO and a subunit β, or to homologues or fragments thereof having similar biological activity, preferably wherein the subunit β is βl. Preferred binding entities are proteins, peptides, carbohydrates, lipids, natural ligands, polyclonal antibodies and monoclonal antibodies. In a further aspect, the invention relates to a fragment of the integrin subunit αlO, which fragment is a peptide chosen from the group comprising peptides of 6 the cytoplasmic domain, the I-domain and the spliced domain.
In one embodiment, said fragment is a peptide comprising the amino acid sequence KLGFFAHKKIPEEEKREEKLEQ. In another embodiment, said fragment comprises the amino acid sequence from about amino acid no. 952 to about amino acid no. 986 of SEQ ID No. 1.
In a further embodiment, said fragment comprises the amino acid sequence from about amino acid No. 140 to about amino acid No. 337 in SEQ ID No. 1.
Another embodiment of the invention relates to a polynucleotide or oligonucleotide coding for a fragment of the human integrin subunit αlO. In one embodiment this polynucleotide of oligonucleotide codes for a fragment which is a peptide chosen from the group comprising peptides of the cytoplasmic domain, the I-domain and the spliced domain. In further embodiments the polynucleotide or oligonucleotide codes for the fragments defined above. The invention also relates to binding entities hav- ing the capability of binding specifically to a fragment of the integrin subunit αlO as defined above.
The invention also relates to a process of using an integrin subunit αlO comprising the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, or an integrin heterodimer comprising said subunit αlO and a subunit β, or a homologue or fragment of said integrin or subunit having similar biological activity, as a marker or target molecule of cells or tissues expressing said integrin subunit αlO, which cells or tissues are of animal includ- ing human origin.
In an embodiment of this process the fragment is a peptide chosen from the group comprising peptides of the cytoplasmic domain, the I-domain and the spliced domain. In further embodiments of said process the frag- ment is a peptide comprising the amino acid sequence KLGFFAHKKIPEEEKREEKLEQ, or a fragment comprising the amino acid sequence from about amino acid No. 952 to 7 about amino acid No. 986 of SEQ ID No. 1, or a fragment comprising the amino acid sequence from about amino acid no. 140 to about amino acid no. 337 of SEQ ID no. 1.
The subunit β is preferably βl. The cells are pre- ferably chosen from the group comprising chondrocytes, smooth muscle cells, endothelial cells, osteoblasts and fibroblasts.
Said process may be used during pathological conditions involving said subunit αlO, such as pathological conditions comprising damage of cartilage, or comprising trauma, rheumatoid arthritis and osteoarthritis .
Said process may be used for detecting the formation of cartilage during embryonal development, or for detecting physiological or therapeutic reparation of cartilage. Said process may also be used for selection and analysis, or for sorting, isolating or purification of chondrocytes.
A further embodiment of said process is a process for detecting regeneration of cartilage or chondrocytes during transplantation of cartilage or chondrocytes.
A still further embodiment of said process is a process for in vitro studies of differentiation of chondrocytes .
The invention also comprises a process of using binding entities having the capability of binding specifically to an integrin subunit αlO comprising the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, or an integrin heterodimer comprising said subunit αlO and a subunit β, or to homologues or fragments thereof having similar biological activity, as markers or target molecules of cells or tissues expressing said integrin subunit αlO, which cells or tissues are of animal including human origin.
The fragment in said process may be a peptide chosen from the group comprising peptides of the cytoplasmic domain, the I-domain and the spliced domain. In preferred embodiments said fragment is a peptide comprising the 8 amino acid sequence KLGFFAHKKIPEEEKREEKLEQ, or a fragment comprising the amino acid sequence from about amino acid No. 952 to about amino acid No. 986 of SEQ ID No. 1, or a fragment comprising the amino acid sequence from about amino acid No. 140 to about amino acid no. 337 of SEQ ID No. 1.
The process may also be used for detecting the presence of an integrin subunit αlO comprising the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, or of an integrin heterodimer comprising said subunit αlO and a subunit β, or of homologues or fragments thereof having similar biological activity.
In a further embodiment said process is a process for determining the differentiation-state of cells during embryonic development, angiogenesis, or development of cancer. '
In a still further embodiment this process is a process for detecting the presence of an integrin subunit αlO, or of a homologue or fragment of said integrin subunit having similar biological activity, on cells, whereby a polynucleotide or oligonucleotide chosen from the group comprising a polynucleotide or oligonucleotide chosen from the nucelotide sequence shown in SEQ ID No. 1 is used as a marker under hybridisation conditions where- in said polynucleotide or oligonucleotide fails to hybridise to a DNA or RNA encoding an integrin subunit αl . Said cells may be chosen from the group comprising chondrocytes, smooth muscle cells, endothelial cells, osteoblasts and fibroblasts. Said integrin fragment may be a peptide chosen from the group comprising peptides of the cytoplasmic domain, the I-domain and the spliced domain, such as a peptide comprising the amino acid sequence KLGFFAHKKIPEEEKREEKLEQ, or a fragment comprising the amino acid sequence from about amino acid no. 952 to about amino acid no. 986 of SEQ ID No. 1, or a fragment comprising the amino acid sequence from about amino acid No. 140 to about amino acid no. 337 of SEQ ID No. 1. 9
In a still further embodiment the process is a pro¬ cess for determining the differentiation-state of cells during development, in pathological conditions, in tissue regeneration or in therapeutic and physiological repara- tion of cartilage. The pathological conditions may be any pathological conditions involving the integrin subunit αlO, such as rheumatoid arthritis, osteoarthrosis or cancer. The cells may be chosen from the group comprising chondrocytes, smooth muscle cells, endothelial cells, osteoblasts and fibroblasts.
The invention also relates to a process for determining the differentiation-state of cells during development, in pathological conditions, in tissue regeneration and in therapeutic and physiological reparation of car- tilage, whereby a polynucleotide or oligonucleotide chosen from the nucleotide sequence shown* in SEQ ID No. 1 is used as a marker under hybridisation conditions wherein said polynucleotide or oligonucleotide fails to hybridise to a DNA or RNA encoding an integrin subunit αl . Embodiments of this aspect comprise a process, whereby said polynucleotide or oligonucleotide is a polynucleotide or oligonucleotide coding for a peptide chosen from the group comprising peptides of the cytoplasmic domain, the I-domain and the spliced domain, such as a polynucleotide or oligonucleotide coding for a peptide comprising the amino acid sequence KLGFFAHKKIPEEEKREEKLEQ, or comprising the amino acid sequence from about amino acid No. 952 to about amino acid no. 986 of SEQ ID No. 1, or the amino acid sequence from about amino acid No. 140 to about amino acid No. 337 of SEQ ID No. 1. Said pathological conditions may be any pathological conditions involving the integrin subunit αlO, such as rheumatoid arthritis, osteoarthrosis or cancer, or atherosclerosis or inflammation. Said cells may be chosen from the group comprising chondrocytes, smooth muscle cells, endothelial cells, osteoblasts and fibroblasts . 10
In a further aspect the invention relates to a pharmaceutical composition comprising as an active ingredient a pharmaceutical agent or an antibody which is capable of using an integrin heterodimer comprising a subunit αlO and a subunit β, or the subunit αlO thereof, or a homo- logue or fragment of said integrin or subunit αlO having similar biological activity, as a target molecule. An embodiment of said pharmaceutical composition is intended for use in stimulating, inhibiting or blocking the forma- tion of cartilage, bone or blood vessels. A further embodiment comprises a pharmaceutical composition for use in preventing adhesion between tendon/ligaments and the surrounding tissue after infection, inflammation and after surgical intervention where adhesion impairs the function of the tissue.
The invention is also related to a vaccine comprising as an active ingredient an integrin heterodimer comprising a subunit αlO and a subunit β, or the subunit αlO thereof, or a homologue or fragment of said integrin or subunit αlO, or DNA or RNA coding for said integrin subunit αlO.
A further aspect of the invention is related to the use of the integrin subunit αlO as defined above as a marker or target in transplantation of cartilage or chon- drocytes.
A still further aspect of the invention is related to a method of using binding entities having the capability of binding specifically to an integrin subunit αlO comprising the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, or an integrin heterodimer comprising said subunit αlO and a subunit β, or to homologues or fragments thereof having similar biological activity, for promoting adhesion of chondrocytes and/or osteoblasts to surfaces of implants to stimulate osseointegration. The invention is also related to the use of an integrin subunit αlO or an integrin heterodimer comprising said subunit αlO and a subunit β as a target for anti- 11 adhesive drugs or molecules in tendon, ligament, skeletal muscle or other tissues where adhesion impairs the function of the tissue.
The invention also relates to a method of stimulat- ing, inhibiting or blocking the formation of cartilage or bone, comprising administration to a subject a suitable amount of a pharmaceutical agent or an antibody which is capable of using an integrin heterodimer comprising a subunit αlO and a subunit β, or the subunit αlO thereof, or a homologue or fragment of said integrin or subunit αlO having similar biological activity, as a target molecule.
In another embodiment the invention is related to a method of preventing adhesion between tendon/ligaments and the surrounding tissue after infection, inflammation and after surgical intervention where adhesion impairs the function of the tissue, comprising administration to a subject a suitable amount of a pharmaceutical agent or an antibody which is capable of using a integrin hetero- dimer comprising a subunit αlO and a subunit β, or the subunit αlO thereof, or a homologue or fragment of said integrin or subunit αlO having similar biological activity, as a target molecule.
The invention also relates to a method of stimulat- ing extracellular matrix synthesis and repair by activation or blockage of an integrin heterodimer comprising a subunit αlO and a subunit β, or of the subunit αlO thereof, or of a homologue or fragment of said integrin or subunit αlO having similar biological activity. In a further aspect the invention relates to a method of in vitro detecting the presence of integrin binding entities, comprising interaction of an integrin heterodimer comprising a subunit αlO and a subunit β, or the subunit αlO thereof, or a homologue or fragment of said integrin or subunit, with a sample, thereby causing said integrin, subunit αlO, or homologue or fragment thereof having similar biological activity, to modulate 12 the binding to its natural ligand or other integrin bind¬ ing proteins present in said sample.
The invention also relates to a method of in vitro studying consequences of the interaction of a human heterodimer integrin comprising a subunit αlO and a subunit β, or the subunit αlO thereof, or a homologue or fragment of said integrin or subunit, with an integrin binding entity and thereby initiate a cellular reaction. Said consequences may be measured as alterations in cel- lular functions.
A still further aspect of the inventions relates to a method of using DNA or RNA encoding an integrin subunit αlO or homologues or fragments thereof as a molecular target. In an embodiment of this aspect, a polynucleotide or oligonucleotide hybridises to the DNA or RNA encoding an integrin subunit αlO or homologues or fragments there¬ of, whereby said polynucleotide or oligonucleotide fails to hybridise to a DNA or RNA encoding en integrin subunit αl. The invention also relates to a method of using a human heterodimer integrin comprising a subunit αlO and a subunit β, or the subunit αlO thereof, or a homologue or fragment of said integrin or subunit, or a DNA or RNA encoding an integrin subunit αlO or homologues or frag- ments thereof, as a marker or target molecule during angiogenesis . BRIEF DESCRIPTION OF THE FIGURES
Fig.l Affinity purification of the αlO integrin subunit on collagen type II-Sepharose. Fig. 2. Amino acid sequences of peptides from the bovine αlO integrin subunit.
Fig. 3a. Affinitypurification and immunoprecipi- tation of the integrin subunit αlO from bovine chondrocytes . Fig. 3b. Affinitypurification and immunoprecipita- tion of the integrin subunit αlO from human chondrocytes. 13
Fig. 3c. Affinitypurification and immunoprecipita- tion of the integrin subunit αlO from human chondrosar- coma cells.
Fig. 4. A 900 bp PCR-fragment corresponding to the bovine integrin subunit αlO
Fig. 5. Schematic map of the three overlapping αlO clones .
Fig. 6. Nucleotide sequence and deduced amino acid sequence of the human αlO integrin subunit. Fig. 7. Northern blot of integrin αlO mRNA.
Fig. 8 Immunoprecipitation of the αlO integrin subunit from human chondrocytes using antibodies against the cytoplasmic domain of αlO (a) . Western blot of the αlO associated β-chain (b) . Fig. 9. Immunostaining of αlO integrin in human articular cartilage. '
Fig. 10 Immunostaining of αlO integrin in 3 day mouse limb cartilage.
Fig 11. Immunostaining of αlO integrin in 13.5 day mouse embryo.
Fig 12. Hybridisation of αlO mRNA in various human tissues.
Fig. 13 Immunostaining of fascia around tendon (a) , skeletal muscle (b) and heart valves (c) in 3 day mouse limb.
Fig. 14. PCR fragments corresponding to αlO integrin subunit from human chondrocytes, human endothelial cells, human fibroblasts and rat tendon.
Fig 15. Partial genomic nucleotide sequence of the human integrin subunit αlO.
Fig 16. Upregulation of αlO integrin subunit in chondrocytes cultured in alginate.
Fig 17. Immunoprecipitation of the αlO integrin subunit from human smooth muscle cells
DETAILED DESCRIPTION OF THE INVENTION
The present invention demonstrate that human and 14 bovine chondrocytes express a novel, collagen type II-binding integrin in the βl-family. An earlier study presented some evidence for that human chondrosarcoma cells also express this integrin (25) . Immunoprecipita- tion experiments using antibodies against the integrin subunit βl revealed that this novel α-integrin subunit had an apparent molecular weight (Mr ) of approximately 160 kDa under reducing conditions, and was slightly larger than the α2 integrin subunit. To isolate this α-subunit collagen type II-binding proteins were affinity purified from bovine chondrocytes. The chondrocyte lysate was first applied to a fibronectin-Sepharose precolumn and the flow through was then applied to a collagen type II-Sepharose column. A protein with Mr of approximately 160 kD was specifically eluted with EDTA from the collagen column but not from the fibronectin cblumn. The Mr of this protein corresponded with the Mr of the unidentified βl-related integrin subunit. The 160 kD protein band was excised from the SDS-PAGE gel, digested with trypsin and the amino acid sequences of the isolated peptides were analysed.
Primers corresponding to isolated peptides amplified a 900 bp PCR-fragment from bovine cDNA which was cloned, sequenced and used for screening of a human articular chondrocyte λZapII cDNA library to obtain the human integrin α-subunit homologue. Two overlapping clones, hcl and hc2 were isolated, subcloned and sequenced. These clones contained 2/3 of the nucleotide sequence including the 3' end of the cDNA. A third clone which contained the 5 ' end of the αlO cDNA, was obtained using the RACE technique. Sequence analysis of the 160 kD protein sequence showed that it was a member of the integrin α-subunit family and the protein was named αlO.
The deduced amino acid sequence of αlO was found to share the general structure of the integrin α-subunits described in previously published reports (6-21) . The large extracellular N-terminal part of αlO contains a 15 seven-fold repeated sequence which was recently predicted to fold into a β-propeller domain (32) . The integrin subunit αlO contains three putative divalent cation-binding sites (DxD/NxD/NxxxD) (53), a single spanning transmem- brane domain and a short cytoplasmic domain. In contrast to most α-integrin subunits the cytoplasmic domain of αlO does not contain the conserved sequence KxGFF (R/K) R. The predicted amino acid sequence in αlO is KLGFFAH. Several reports indicate that the integrin cytoplasmic domains are crucial in signal transduction (54) and that membrane-proximal regions of both α- and β-integrin cytoplasmic domains are involved in modulating conformation and affinity state of integrins (55-57) . It is suggested that the GFFKR motif in α-chains are important for asso- ciation of integrin subunits and for transport of the integrin to the plasma membrane (58) . The* KxGFFKR domain has been shown to interact with the intracellular protein calreticulin (59) and interestingly, calreticulin-null embryonic stem cells are deficient in integrin-mediated cell adhesion (60) . It is therefor possible that the sequence KLGFFAH in αlO have a key function in regulating the affinity between αlOβl and matrix proteins.
Integrin α subunits are known to share an overall identity of 20-40% (61) . Sequence analysis showed that the αlO subunit is most closely related to the I-domain containing α-subunits with the highest identity to αl (37%) and α2 (35%) . The integrins αlβl and α2βl are known receptors for both collagens and laminins (24; 62; 63) and we have also recently demonstrated that α2βl interacts with the cartilage matrix protein chondroadherin (42) .
Since αlOβl was isolated on a collagen type II-Sepharose, we know that collagen type II is a ligand for αlOβl. We have also shown by affinity purification experiments that αlOβl interacts with collagen type I but it remains to be seen whether laminin or chondroadherin are also ligands for this integrin. 16
The αlO associated β-chain migrated as the βl integrin subunit both under reducing and non-reducing conditions. To verify that the αlO associated β-chain indeed is βl, chondrocyte lysates were immunoprecipitated with antibodies against αlO or βl followed by Western blot using antibodies against the βl-subunit. These results clearly demonstrated that αlO is a member of the βl-integrin family. However, the possibility that αlO combine also with other β-chains can not be excluded.. A polyclonal peptide antibody raised against the cytoplasmic domain of αlO precipitated two protein bands with Mr of approximately 160 kD (αlO) and 125 kD (βl) under reducing conditions. Immunohistochemistry using the αlO-antibody showed staining of the chondrocytes in tis- sue sections of human articular cartilage. The antibody staining was clearly specific since preincubation of the antibody with the αlO-peptide completely abolished the staining. Immunohistochemical staining of mouse limb sections from embryonic tissue demonstrated that αlO is upregulated during condensation of the mesenchyme. This indicate that the integrin subunit αlO is important during the formation of cartilage. In 3 day old mice αlO was found to be the dominating collagen binding integrin subunit which point to that αlO has a key function in maintaining normal cartilage functions.
Expression studies on the protein and mRNA level show that the distribution of αlO is rather restrictive. Immunohistochemistry analyses have shown that αlO integrin subunit is mainly expressed in cartilage but it is also found in perichondrium, periosteum, ossification groove of Ranvier, in fascia surrounding tendon and skeletal muscle and in the tendon-like structures in the heart valves. This distribution point to that αlO integrin subunit is present also on fibroblasts and osteoblasts. PCR amplification of cDNA from different cell types revealed the presence of an alternatively spliced αlO integrin subunit. This spliced αlO was domi- 17 nating in fibroblasts which suggests that αlO in fibroblasts may have a different function compared to αlO present on chondrocytes.
Expression of the integrin subunit αlO was found to decrease when chondrocytes were cultured in monolayer. In contrast, the expression of αlO was found to increase when the cells were cultured in alginate beads. Since the latter culturing model is known to preserve the phenotype of chondrocytes the results suggest that αlO can function as marker for a differentiated chondrocyte.
Adhesion between tendon/ligaments and the surround¬ ing tissue is a well-known problem after infection, injury and after surgical intervention. Adhesion between tendon and tendon sheets impairs the gliding function and cause considerable problems especially during healing of tendons in e.g. the hand and fingers leading to functional incapacity. The localisation of the αlO integrin subunit in the fascia of tendon and skeletal muscle makes αlO a possible target for drugs and molecules with anti- adhesive properties that could prevent impairment of the function of tendon/ligament. The integrin subunit αlO can also be a target for drugs or molecules with anti-adhesive properties in other tissues where adhesion is a problem.
EXAMPLES
Example 1
Affinity purification of the αlOintegrin subunit on collagen type II-Sepharose. Materials and Methods
Bovine chondrocytes, human chondrocytes or human chondrosarcoma cells were isolated as described earlier [Holmvall et al, Exp Cell Res, 221, 496-503 (1995), Camper et al, JBC, 273, 20383-20389 (1998)]. A Triton X-100 lysate of bovine chondrocytes was applied to a fibronectin-Sepharose precolumn followed by a collagen 18 type Il-Sepharose column and the integrin subunit αlO was eluted from the collagen type II-column by EDTA (Camper et al, JBC, 273, 20383-20389 (1998) . The eluted proteins were precipitated by methanol/chloroform, separated by SDS-PAGE under reducing conditions and stained with Coomassie blue. (Camper et al, JBC, 273, 20383-20389 (1998) . Peptides from the αlO protein band were isolated by in-gel digestion with a trypsin and phase liquid chro- matography and sequenced by Edman degradation (Camper et al, JBC, 273, 20383-20389 (1998). Results
Fig 1 shows EDTA-eluted proteins from the fibronec- tin-Sepharose (A) , flow-through from the collagen type Il-Sepharose column (B) and EDTA-eluted proteins from the collagen type Il-Sepharose (C) . The αlO integrin subunit (160 kDa) which was specifically eluted from the collagen type II column is indicated with an arrow. Figure 2 shows the amino acid sequences of six peptides that were isolated from the bovine integrin subunit αlO. Figures 3 a, b, and c show that the αlO integrin subunit is present on bovine chondrocytes (3a) , human chondrocytes (3b) and human chondrosarcoma cells (3c) . The affinity for collagen type II, the coprecipitation with βl-integrin subunit and the molecular weight of 160 kDa under reducing condi- tions identify the αlO integrin subunit on the different cells. These results show that αlO can be isolated from chondrocytes and from chondrosarcoma cells.
Example 2 Amplification of PCR fragment corresponding to bovine αlO integrin subunit. Materials and methods
The degenerate primers GAY AAY ACI GCI CAR AC (DNTAQT, forward) and TIA TIS WRT GRT GIG GYT (EPHHSI, reverse) were used in PCR (Camper et al, JBC, 273, 20383- 20389 (1998) to amplify the nucleotide sequence corresponding to the bovine peptide 1 (Figure 2) . A 900 bp 19
PCR-fragment was then amplified from bovine cDNA using an internal specific primer TCA GCC TAC ATT CAG TAT (SAYIQY, forward) corresponding to the cloned nucleotide sequence of peptide 1 together with the degenerate primer ICK RTC CCA RTG ICC IGG (PGHWDR, reverse) corresponding to the bovine peptide 2 (Figure2) . Mixed bases were used in positions that were twofold degenerate and inosines were used in positions that are three- or fourfold degenerate. mRNA isolation and cDNA synthesis was done as earlier described (Camper et al, JBC, 273, 20383-20389 (1998)). The purified fragment was cloned, purified and sequenced as earlier described (Camper et al, JBC, 273, 20383-20389 (1998) ) . Results The nucleotide sequence of peptide 1 (Figure 2) was obtained by PCR-amplification, clonin'g and sequencing of bovine cDNA. From this nucleotide sequence an exact primer was designed and applied in PCR-amplification with degenerate primers corresponding to peptides 2-6 (Figure 2) . Primers corresponding to peptides 1 and 2 amplified a 900 bp PCR-fragment from bovine cDNA (Figure 4) .
Example 3 Cloning and sequence analysis of the human αlO integrin subunit Material and methods
The cloned 900bp PCR-fragment, corresponding to bovine αlO-integrin, was digoxigenin-labelled according to the DIG DNA labelling kit (Boehringer Mannheim) and used as a probe for screening of a human articular chondrocyte λZapII cDNA library (provided by Michael Bayliss, The Royal Veterinary Basic Sciences, London, UK) (52) . Positive clones containing the pBluescript SK+ plasmid with the cDNA insert were rescued from the ZAP vector by in vivo excision as described in the ZAP-cDNA® synthesis kit (Stratagene) . Selected plasmids were purified and 20 sequenced as described earlier (Camper et al, JBC, 273, 20383-20389 (1998)) using T3, T7 and internal specific primers. To obtain cDNA that encoded the 5' end of αlO we designed the primer AAC TCG TCT TCC AGT GCC ATT CGT GGG (reverse; residue 1254-1280 in αlO cDNA) and used it for rapid amplification of the cDNA 5' end (RACE) as described in the Marathon™ cDNA Amplification kit (Clontech INC. , Palo Alto, CA) . Results Two overlapping clones, hcl and hc2 (Figure 5), were isolated, subcloned and sequenced. These clones contained 2/3 of the nucleotide sequence including the 3' end of the cDNA. A third clone (racel; Figure 5), which contained the 5 ' end of the αlO cDNA, was obtained using the RACE technique. From these three overlapping clones of αlO cDNA, 3884 nucleotides were sequenced4 The nucleotide sequence and deduced amino acid sequence is shown in Figure 6. The sequence contains a 3504-nucleotide open reading frame that is predicted to encode a 1167 amino acid mature protein. The signal peptide cleavage site is marked with an arrow, human homologues to bovine peptide sequences are underlined and the I-domain is boxed. Metal ion binding sites are indicated with a broken underline, potential N-glycosylation sites are indicated by an asterisk and the putative transmembrane domain is double underlined. The normally conserved cytoplasmic sequence is indicated by a dot and dashed broken underline.
Sequence analysis demonstrate that αlO is a member of the integrin α-subunit family.
Example 4
Identification of a clone containing a splice variant of αlO
One clone which was isolated from the human chon- drocyte library (see Example 3) contained a sequence that was identical to the sequence of αlO integrin subunit except that the nucleotides between nt positions 21
2942 and 3055 were deleted. The splice variant of αlO was verified in PCR experiment using primers flanking the splice region (see figure 14).
Example 5
Identification of αlO integrin subunit by Northern blot Material and methods
Bovine chondrocyte mRNA was purified using a QuickPrep®Micro mRNA Purification Kit (Pharmacia Biotech, Uppsala, Sweden) , separated on a 1% agarose-formaldehyde gel, transferred to nylon membranes and immobilised by UV crosslinking. cDNA-probes were 32P-labelled with Random Primed DNA Labeling Kit (Boehringer Mannheim) . Filters were prehybridised for 2-4 hours at 42°C in 5x SSE,
5x Denharts solution, 0.1 % SDS, 50 μg/ml* salmon sperm DNA and 50% formamide and then hybridised over night at 42 °C with the same solution containing the specific probe (0.5-1 x 106 cpm/ml) . Specifically bound cDNA- probes were analysed using the phosphoimager system (Fuji). Filters were stripped by washing in 0.1% SDS, for 1 hour at 80°C prior to re-probing. The αlO-integrin cDNA-probe was isolated from the racel-containing plasmid using the restriction enzymes BamHI (GIBCO BRL) and Ncol (Boehringer Mannheim) . The rat βl-integrin cDNA probe was a kind gift from Staffan Johansson, Uppsala, Sweden. Results
Northern blot analysis of mRNA from bovine chondrocytes showed that a human αlO cDNA-probe hybridised with a single mRNA of approximately 5.4 kb (Figure 7) . As a comparison, a cDNA-probe corresponding to the integrin subunit αl was used. This cDNA-probe hybridised a RNA- band of approximately 3.5 kb on the same filter. These results show that a cDNA-probe against αlO can be used to identify the αlO integrin subunit on the mRNA level. 22
Example 6
Preparation of antibodies against the integrin subunit αlO
A peptide corresponding to part of the αlO cytoplas- mic domain, Ckkipeeekreekle (see figure 6) was synthesis- ed and conjugated to keyhole limpet hemocyanin (KLH) . Rabbits were immunised with the peptide-KLH conjugate to generate antiserum against the integrin subunit αlO. Antibodies recognising αlO were affinity purified on an peptide-coupled column (Innovagen AB) .
Example 7
Immunoprecipitation of the integrin subunit αlO from chondrocytes Material and methods
Human chondrocytes were 125I-labelled*, lyzed with Triton X-100 and immunoprecipitated as earlier described (Holmvall et al, Exp Cell Res, 221, 496-503 (1995), Camper et al, JBC, 273, 20383-20389 (1998)). Triton X-100 lysates of 1251-labeled human chondrocytes were immunoprecipitated with polyclonal antibodies against the integrin subunits βl, αl, α2, α3 or αlO. The immunoprecipitated proteins were separated by SDS-PAGE (4-12%) under non-reducing conditions and visualised using a phospho- imager. Triton X-100 lysates of human chondrocytes immunoprecipitated with αlO or βl were separated by SDS-PAGE (8%) under non-reducing conditions and analysed by Western blot using the polyclonal βl antibody and chemi- luminescent detection as described in Camper et al, JBC, 273, 20383-20389 (1998). Results
The polyclonal peptide antibody, raised against the cytoplasmic domain of αlO, precipitated two protein bands with Mr of approximately 160 kD (αlO) and 125 kD (βl) under reducing conditions. The αlO associated β-chain migrated as the βl integrin subunit (Figure 8a) . To verify that the αlO associated β-chain in chondrocytes 23 indeed is βl, chondrocyte lysates were immunoprecipitated with antibodies against αlO orb βl followed by Western blot using antibodies against the βl-subunit (Figure 8b) . These results clearly demonstrated that αlO is a member of the βl-integrin family. However, the results do not exclude the possibility that αlO can associate with other β-chains in other situations.
Example 8 Immunohistochemical staining of the integrin subunit αlO in human and mouse cartilage Material and methods
Frozen sections of adult cartilage (trochlear groove) obtained during surgery (provided by Anders Lindahl, Salgrenska Hospital, Gothenburg, Sweden and frozen sections from of 3 day old mouse limb were fixed and prepared for immunohistochemistry as earlier described (Camper et al, JBC, 273, 20383-20389 (1998)). Expression of αlO integrin subunit was analysed using the poly- clonal antibody against the cytoplasmic domain as a primary antibody (see Example 6) and a secondary antibody conjugated to peroxidase. Results
Figures 9 show immunostaining of human adult articu- lar cartilage.
The αlO-antibody recognising the cytoplasmic domain of αlO stained the chondrocytes in tissue sections of human articular1 cartilage (A) . The staining was depleted when the antibody was preincubated with the αlO- peptide (B) . A control antibody recognising the α9 integrin subunit did not bind to the chondrocyte (C) .
Figures 10 shows ithat the αlO antibody stain the majority of chondrocytes in the growing bone anlage (a and b) . The αlO antibody also recognised cells in the ossification groove of Ranvier (b) , especially the osteoblast in the bone bark which are lining the cartilage in the metaphys are highly positive for αlO. The 24 cells in the ossification groove of Ranvier are believed to be important for the growth in diameter of the bone. The integrin subunit αlO is also highly expressed in perichondrium and periosteum. Cell in these tissues are likely important in the repair of the cartilage tissue. The described localisation of the integrin subunit αlO suggest that this integrin is important for the function of the cartilage tissue.
Example 9
Immunohistochemical staining of the integrin subunit αlO during mouse development Material and methods
Frozen sections from mouse embryos (13.5 days) were investigated for expression of αlO by immunhistochemi- stry as described in Camper et al, JBC, 273, 20383-20389 (1998). Expression of αlO integrin subunit was analysed using the polyclonal antibody against the cytoplasmic domain as a primary antibody (see Example 6) and a secon- dary antibody conjugated to peroxidase. The embryo sections were also investigated for expression of integrin subunit αl (monoclonal antibody from Pharmingen) and collagen type II (monoclonal antibody, kind gift from Dr John Mo, Lund University, Sweden) . Results
Figure 11 show that αlO integrin subunit is unregulated in the limb when the mesenchymal cells undergo condensation to form cartilage (a) . Especially the edge of the newly formed cartilage has high expression of αlO. The formation of cartilage is verified by the high expression of the cartilage specific collage type II (b) . The control antibody against αl integrin subunit showed only weak expression on the cartilage (c) . In other experiments expression of :αl0 was found in all cartilage con- taining tissues in the 3 day old mouse including limbs, ribs and vertebrae. The upregulation of αlO during formation of cartilage suggest that this integrin subunit is 25 important both in the development of cartilage and bone and in the repair of damaged cartilage tissue.
Example 10 mRNA expression of αlO in tissues other than articu¬ lar cartilage Material and methods
Expression of αlO integrin subunit was examined on the mRNA level in different human tissues. A Northern dot blot with immobilised mRNA from the listed tissues in
Figure 12 was hybridised with an αlO integrin cDNA probe isolated from the race 1-containing plasmid using the restriction enzymes BamHl and col . The degree of hybridisation was analysed using a phospho imager. The follow- ing symbols denote mRNA level in increasing order: -, +, ++, +++, ++++. '
Results
Analysis of the hybridised mRNA showed that αlO was expressed in aorta, trachea, spinal cord, heart, lung, and kidney (Figure 12) . All other tissues appeared negative for αlO expression. These results point to a restricted distribution of the αlO integrin subunit.
Example 11 Immunohistochemical staining of αlO in fascia around tendon and skeletal muscle and in tendon structures in heart valves. Materials and methods
Frozen sections of adult cartilage (trochlear groove) obtained during surgery (provided by Anders Lindahl, Salgrenska Hospital, Gothenburg, Sweden and frozen sections from of 3 day old mouse limb were fixed and prepared for immunohistochemistry as earlier described (Camper et al, JBC, 273, 20383-20389 (1998)). Expres- sion of αlO integrin subunit was analysed using the polyclonal antibody against the cytoplasmic domain as a pri- 26 mary antibody (see Example 6) and a secondary antibody conjugated to peroxidase.
Results
As shown in figures 13 expression of αlO was found in the fascia surrounding tendon (a) and skeletal muscle (b) and in the tendon structures in the heart valves (c) . This localisation suggest that αlO can bind to other matrix molecules in addition to the cartilage specific collagen type II. The localisation of the integrin αlO on the surface of tendons indicate that αlO can be involved in unwanted adhesion that often occurs between tendon/ ligaments and the surrounding tissue after infection, injury or after surgery.
Example 12 mRNA expression of αlO integrin subuhit in chondrocytes, endothelial cells and fibroblasts. Material and methods
Isolation of mRNA, synthesis of cDNA and PCR ampli- fication was done as earlier described (Camper et al, JBC, 273, 20383-20389 (1998)). Results
Figure 14- shows PCR amplification of αlO cDNA from human articular chondrocytes (lanes A6 and Bl) , human umbilical vein endothelial cells (lane A2) , human fibroblasts (lane A4) and rat tendon (Fig 14b, lane B2) . Lanes 1, 3, and 5 in figure 14 A show amplified fragments corresponding to the integrin subunit α2 in endothelial cells, fibroblasts and chondrocytes, respectively. cDNA- primers corresponding to the αlO sequence positions nt 2919-2943 (forward) and nt 3554-3578 (reverse) (see Figure 6) were used to amplify αlO cDNA from the different cells. The figure shows that αlO was amplified in all three cell types. Two fragments of αlO was amplified which represent the intact form of αlO (larger fragment) and a splice variant (smaller fragment) . The larger frag- 27 ment was dominating in chondrocytes while the smaller fragment was more pronounced in tendon (B2) .
Example 13 Construction of αlO mammalian expression vector.
The full length protein coding sequence of αlO (combined from 3 clones, see figure 6) was inserted into the mammalian expression vector, pcDNA3.1/Zeo ( Invitrogen) . The vector contains SV40 promoter and Zeosin selection sequence. The αlO containing expression vector was trans- fected into cells that express the βl-integrin subunit but lack expression of the αlO subunit. Expression of the αlO integrin subunit on the cell surface can be analysed by immunoprecipitation and/or flow cytometry using anti- bodies specific for αlO. The ligand binding capacity and the function of the inserted αlO integrin* subunit can be demonstrated in cell adhesion experiment and in signalling experiments.
Example 14
Construction of mammalian expression vector contain¬ ing a splice variant of αlO.
The full length protein coding sequence of the splice variant of αlO (nt 2942-nt3055 deleted) was inserted into the mammalian expression vector pcDNA3
(see Example 13) . Expression and function of the splice variant can be analysed as described in example 13 and compared with the intact αlO integrin subunit.
Example 15
Partial isolation and characterisation of the αlO integrin genomic DNA Material and methods
Human αlO cDNA, isolated from the racel-containing plasmid using the restriction enzymes BamRI (GIBCO BRL) and Ncol (Boehringer Mannheim) , was 32P-labelled and used as a probe for screening of a mouse 129 cosmid library 28
(provided by Reinhard Fassler, Lund University) . Positive clones were isolated and subcloned. Selected plasmids were purified and sequenced as described earlier (Camper et al, JBC, 273, 20383-20389 (1998)) using T3, T7 and internal specific primers. Primers corresponding to mouse genomic DNA were then constructed and used in PCR to amplify and identify the genomic sequence of αlO from the cosmid clones. Results Figure 15 shows 7958 nt of the αlO gene. This partial genomic DNA sequence of αlO integrin contains 8 exons, and a Kozak sequence. The mouse genomic αlO sequence was used to generate a targeting vector for knockout experiments.
Example 16
Upregulation of αlO integrin subunit in chondrocytes cultured in alginate beads Material and methods ; Human chondrocytes cultured in monolayer for 2 weeks were detached with trypsin-EDTA and introduced into alginate beads. Chondrocytes cultured in alginate are known to preserve their phenotype while chondrocytes cultured in monolayer are dedifferentiated. After 11 days chondro- cytes cultured either in alginate or on monolayer were isolated and surface labelled with 125I. The αlO integrin subunit was then immunoprecipitated with polyclonal antibodies recognising the cytoplasmic domain of αlO (see Example 6 and Camper et al, JBC, 273, 20383-20389 (1998)). Results
As shown in figure 16 chondrocytes cultured in alginate beads (lanes 3 and 4) upregulated their protein expression of αlOβl. This was in contrast to chondrocytes cultured in monolayer (lanes 1 and 2) which had a very low expression of αlOβl. Immunoprecipitation with ab control antibody is shown in lanes 1 and 3. It is known that 29 chondrocytes preserve their cartilage specific matrixpro- duction in alginate cultures but not in monolayer culture which point to that alginate preserve the phenotype of chondrocytes. These results support that αlO integrin subunit can be used as a marker for differentiated chondrocytes .
Example 17
Immunoprecipitation of the αlO integrin subunit from human smooth muscle cells.
Material and methods
Human smooth muscle cells were isolated from human aorta. After one week in culture the cells were 1 5l- labelled, lysed and immunoprecipitated with antibodies against the integrin subunit βl (lane 1), αl (lane 2), α2
(lane 3), αlO (lane 4), α3 (lane 5), control (lane 6)
(Figure 17) . The experiment was done as described in
Example 7.
Results The αlO antibody precipitated two bands from the smooth muscle cells corresponding to the αlO and the βl integrin subunit (Fig. 17) .
Example 18 Construction of bacterial expression vector containing sequence for αlO splice region.
A plasmid for intracellular expression in E. coli of the alternatively spliced region (amino acid pos . 952-986, SEQ. ID 1) was constructed as described. The alternatively spliced region were back-translated using the E. coli high frequency codon table, creating a cDNA sequence of 96% identity with the original sequence (SEQ. ID 1 nucleotide pos 2940-3044). Using sequence overlap extension (Horton et al., Biotechniques 8:528, 1990) primer αlOpfor (tab. I) and αlOprev (tab. I) was used to generate a double stranded fragment encoding the αlO amino acid sequence. This fragment was used as a PCR 30 template with primers αl0pfor2 (tab. I) and αl0prev2 (tab. I) in order to generate restriction enzyme site for sub-cloning in a pET vector containing the Z-domain of staphylococcal protein A, creating a fusion of the αlO spliced region with the amino terminal of the Z-domain with trombin cleavage site residing in-between. The fragment generated in the second PCR reaction is shown (SEQ ID No. 3) also indicating the unique restriction enzymes used for sub-cloning in the expression vector.
Table I αlOpfor 5'-
GTTCAGAACCTGGGTTGCTACGTTGTTTCCGGTCTGATCATCTCCGC TCTGCTGCCGGCTGT-3' αl0pfor2 5' -GGGGCATATGGTTCAGAACCTGGGTTGCTACGTTG-3'
αlOprev 5'- ( ,
GATAACCTGGGACAAGCTTAGGAAGTAGTTACCACCGTGAGCAACAG CCGGCAGCAGAGCGGA-3' αl0prev2 5'-
GGGGGGATCCGCGCGGCACCAGGCCGCTGATAACCTGGGACAAGCTT
AGGAAGT-3'
Figure imgf000032_0001
31
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36 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) NUMBER OF SEQUENCES: 2
(2) INFORMATION FOR SEQ ID NO. 1: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3884 base pairs
(B) TYPE: nucleic acid and amino acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULAR TYPE: cDNA (vi) ORIGINAL SOURCE:
(E) ORGANISM: human
(F) CELLTYPE: chondrocyte
(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 1:
CAGGTCAGAAACCGATCAGGCATGGAACTCCCCTTCGTCACTCACCTGTTCTTGCCCCTG
GTCCAGTCTTTGGCTAGTCCGTACCTTGAGGGGAAGCAGTGAGTGGACAAGAACGGGGAC a M E L P F V T H L F L P L -
GTGTTCCTGACAGGTCTCTGCTCCCCCTTTAACCTGGATGAACATCACCCACGCCTATTC
61 + + + + + + 120
CACAAGGACTGTCCAGAGACGAGGGGGAAATTGGACCTACTTGTAGTGGGTGCGGATAAG a V F L T G L C S P F N L D E H H P R L F
CCAGGGCCACCAGAAGCTGAATTTGGATACAGTGTCTTACAACATGTTGGGGGTGGACAG GGTCCCGGTGGTCTTCGACTTAAACCTATGTCACAGAATGTTGTACAACCCCCACCTGTC a P G P P E A E F G Y S V L Q H V G G G Q
CGATGGATGCTGGTGGGCGCCCCCTGGGATGGGCCTTCAGGCGACCGGAGGGGGGACGTT GCTACCTACGACCACCCGCGGGGGACCCTACCCGGAAGTCCGCTGGCCTCCCCCCTGCAA a R W M V G A P W D G P S G D R R G D V
TATCGCTGCCCTGTAGGGGGGGCCCACAATGCCCCATGTGCCAAGGGCCACTTAGGTGAC
241 + + + + + + 300
ATAGCGACGGGACATCCCCCCCGGGTGTTACGGGGTACACGGTTCCCGGTGAATCCACTG a Y R C P V G G A H N A P C A K G H L G D
TACCAACTGGGAAATTCATCTCATCCTGCTGTGAATATGCACCTGGGGATGTCTCTGTTA
301 + + + + + + 360
ATGGTTGACCCTTTAAGTAGAGTAGGACGACACTTATACGTGGACCCCTACAGAGACAAT a Y Q L G N S S H P A V N M H L G M S L L
GAGACAGATGGTGATGGGGGATTCATGGCCTGTGCCCCTCTCTGGTCTCGTGCTTGTGGC
361 + + + + + + 420
CTCTGTCTACCACTACCCCCTAAGTACCGGACACGGGGAGAGACCAGAGCACGAACACCG a E T D G D G G F M A C A P K S R A C G 37
AGCTCTGTCTTCAGTTCTGGGATATGTGCCCGTGTGGATGCTTCATTCCAGCCTCAGGGA
421 + + + + + + 480
TCGAGACAGAAGTCAAGACCCTATACACGGGCACACCTACGAAGTAAGGTCGGAGTCCCT
S S V F S S G I C A R V D A S F Q P Q G
AGCCTGGCACCCACTGCCCAACGCTGCCCAACATACATGGATGTTGTCATTGTCTTGGAT
TCGGACCGTGGGTGACGGGTTGCGACGGGTTGTATGTACCTACAACAGTAACAGAACCTA
S L A P T A Q R C P T Y M D V V I V L D
GGCTCCAACAGCATCTACCCCTGGTCTGAAGTTCAGACCTTCCTACGAAGACTGGTAGGG
CCGAGGTTGTCGTAGATGGGGACCAGACTTCAAGTCTGGAAGGATGCTTCTGACCATCCC
G S N S I Y P S E V Q T F L R R L V G
AAACTGTTTATTGACCCAGAACAGATACAGGTGGGACTGGTACAGTATGGGGAGAGCCCT
TTTGACAAATAACTGGGTCTTGTCTATGTCCACCCTGACCATGTCATACCCCTCTCGGGA
K L F I D P E Q I Q V G L V Q Y G E S P
GTACATGAGTGGTCCCTGGGAGATTTCCGAACGAAGGAAGAAGTGGTGAGAGCAGCAAAG
CATGTACTCACCAGGGACCCTCTAAAGGCTTGCTTCCTTCTTCACCACTCTCGTCGTTTC
V H E S L G D F R T K E E V V R A A K
AACCTCAGTCGGCGGGAGGGACGAGAAACAAAGACTGCCCAAGCAATAATGGTGGCCTGC
TTGGAGTCAGCCGCCCTCCCTGCTCTTTGTTTCTGACGGGTTCGTTATTACCACCGGACG
N L S R R E G R E T K T A Q A I M V A C
ACAGAAGGGTTCAGTCAGTCCCATGGGGGCCGACCCGAGGCTGCCAGGCTACTGGTGGTT
TGTCTTCCCAAGTCAGTCAGGGTACCCCCGGCTGGGCTCCGACGGTCCGATGACCACCAA
T E G F S Q S H G G R P E A A R L L V V
GTCACTGATGGAGAGTCCCATGATGGAGAGGAGCTTCCTGCAGCACTAAAGGCCTGTGAG
CAGTGACTACCTCTCAGGGTACTACCTCTCCTCGAAGGACGTCGTGATTTCCGGACACTC
V T D G E S H D G E E L P A A L K A C E
GCTGGAAGAGTGACACGCTATGGGATTGCAGTCCTTGGTCACTACCTCCGGCGGCAGCGA
CGACCTTCTCACTGTGCGATACCCTAACGTCAGGAACCAGTGATGGAGGCCGCCGTCGCT
A G R V T R Y G I A V L G H Y L R R Q R
GATCCCAGCTCTTTCCTGAGAGAAATTAGAACTATTGCCAGTGATCCAGATGAGCGATTC
CTAGGGTCGAGAAAGGACTCTCTTTAATCTTGATAACGGTCACTAGGTCTACTCGCTAAG
D P S S F L R E I R T I A S D P D E R F
TTCTTCAATGTCACAGATGAGGCTGCTCTGACTGACATTGTGGATGCACTAGGAGATCGG
1021 + + + + + + 1080
AAGAAGTTACAGTGTCTACTCCGACGAGACTGACTGTAACACCTACGTGATCCTCTAGCC
F F N V T D E A A L T D I V D A L G D R 38
ATTTTTGGCCTTGAAGGGTCCCATGCAGAAAACGAAAGCTCCTTTGGGCTGGAAATGTCT
1081 + + + + + + 1140
TAAAAACCGGAACTTCCCAGGGTACGTCTTTTGCTTTCGAGGAAACCCGACCTTTACAGA
I F G L E G S H A E N E S S F G E M S
CAGATTGGTTTCTCCACTCATCGGCTAAAGGATGGGATTCTTTTTGGGATGGTGGGGGCC 1141 + + + + + + 1200
GTCTAACCAAAGAGGTGAGTAGCCGATTTCCTACCCTAAGAAAAACCCTACCACCCCCGG
Q I G F S T H R K D G I L F G M V G A
TATGACTGGGGAGGCTCTGTGCTATGGCTTGAAGGAGGCCACCGCCTTTTCCCCCCACGA
ATACTGACCCCTCCGAGACACGATACCGAACTTCCTCCGGTGGCGGAAAAGGGGGGTGCT
Y D G G S V L L E G G H R L F P P R
ATGGCACTGGAAGACGAGTTCCCCCCTGCACTGCAGAACCATGCAGCCTACCTGGGTTAC
TACCGTGACCTTCTGCTCAAGGGGGGACGTGACGTCTTGGTACGTCGGATGGACCCAATG
M A L E D E F P P A L Q N H A A Y L G Y
TCTGTTTCTTCCATGCTTTTGCGGGGTGGACGCCGCCTGTTTCTCTCTGGGGCTCCTCGA
AGACAAAGAAGGTACGAAAACGCCCCACCTGCGGCGGACAAAGAGA'GACCCCGAGGAGCT
S V S S M L L R G G R R L F L S G A P R
TTTAGACATCGAGGAAAAGTCATCGCCTTCCAGCTTAAGAAAGATGGGGCTGTGAGGGTT
AAATCTGTAGCTCCTTTTCAGTAGCGGAAGGTCGAATTCTTTCTACCCCGACACTCCCAA
F R H R G K V I A F Q L K K D G A V R V
GCCCAGAGCCTCCAGGGGGAGCAGATTGGTTCATACTTTGGCAGTGAGCTCTGCCCATTG
1441 + + + + + + 1500
CGGGTCTCGGAGGTCCCCCTCGTCTAACCAAGTATGAAACCGTCACTCGAGACGGGTAAC
A Q S L Q G E Q I G S Y F G S E L C P L
GATACAGATAGGGATGGAACAACTGATGTCTTACTTGTGGCTGCCCCCATGTTCCTGGGA
CTATGTCTATCCCTACCTTGTTGACTACAGAATGAACACCGACGGGGGTACAAGGACCCT
D T D R D G T T D V L L V A A P M F L G
CCCCAGAACAAGGAAACAGGACGTGTTTATGTGTATCTGGTAGGCCAGCAGTCCTTGCTG
1561 + + + + + + 1620
GGGGTCTTGTTCCTTTGTCCTGCACAAATACACATAGACCATCCGGTCGTCAGGAACGAC
P Q N K E T G R V Y V Y L V G Q Q S L L
ACCCTCCAAGGAACACTTCAGCCAGAACCCCCCCAGGATGCTCGGTTTGGCTTTGCCATG
1621 + + + + + + 1680
TGGGAGGTTCCTTGTGAAGTCGGTCTTGGGGGGGTCCTACGAGCCAAACCGAAACGGTAC
T L Q G T Q P E P P Q D A R F G F A M
GGAGCTCTTCCTGATCTGAACCAAGATGGTTTTGCTGATGTGGCTGTGGGGGCGCCTCTG
1681 + + + + + + 1740
CCTCGAGAAGGACTAGACTTGGTTCTACCAAAACGACTACACCGACACCCCCGCGGAGAC
G A L P D L N Q D G F A D V A V G P L 39
GAAGATGGGCACCAGGGAGCACTGTACCTGTACCATGGAACCCAGAGTGGAGTCAGGCCC
1741 + + + + + + 1800
CTTCTACCCGTGGTCCCTCGTGACATGGACATGGTACCTTGGGTCTCACCTCAGTCCGGG
E D G H Q G A L Y L Y H G T Q S G V R P
CATCCTGCCCAGAGGATTGCTGCTGCCTCCATGCCACATGCCCTCAGCTACTTTGGCCGA
1801 + + + + + + 1860
GTAGGACGGGTCTCCTAACGACGACGGAGGTACGGTGTACGGGAGTCGATGAAACCGGCT
H P A Q R I A A A S M P H A L S Y F G R
AGTGTGGATGGTCGGCTAGATCTGGATGGAGATGATCTGGTCGATGTGGCTGTGGGTGCC
1861 + + + + + + 1920
TCACACCTACCAGCCGATCTAGACCTACCTCTACTAGACCAGCTACACCGACACCCACGG
S V D G R D L D G D D L V D V A V G A
CAGGGGGCAGCCATCCTGCTCAGCTCCCGGCCCATTGTCCATCTGACCCCATCACTGGAG
GTCCCCCGTCGGTAGGACGAGTCGAGGGCCGGGTAACAGGTAGACTGGGGTAGTGACCTC
Q G A A I L L S S R P I V H L T P S L E
GTGACCCCACAGGCCATCAGTGTGGTTCAGAGGGACTGTAGGCGGCGAGGCCAAGAAGCA
CACTGGGGTGTCCGGTAGTCACACCAAGTCTCCCTGACATCCGCCGfcTCCGGTTCTTCGT
V T P Q A I S V V Q R D C R R R G Q E A
GTCTGTCTGACTGCAGCCCTTTGCTTCCAAGTGACCTCCCGTACTCCTGGTCGCTGGGAT
CAGACAGACTGACGTCGGGAAACGAAGGTTCACTGGAGGGCATGAGGACCAGCGACCCTA
V C T A A L C F Q V T S R T P G R W D
CACCAATTCTACATGAGGTTCACCGCATCACTGGATGAATGGACTGCTGGGGCACGTGCA
2101 + + + + + + 2160
GTGGTTAAGATGTACTCCAAGTGGCGTAGTGACCTACTTACCTGACGACCCCGTGCACGT
H Q F Y M R F T A S L D E T A G A R A
GCATTTGATGGCTCTGGCCAGAGGTTGTCCCCTCGGAGGCTCCGGCTCAGTGTGGGGAAT
2161 + + + + + + 2220
CGTAAACTACCGAGACCGGTCTCCAACAGGGGAGCCTCCGAGGCCGAGTCACACCCCTTA
A F D G S G Q R L S P R R L R L S V G N
GTCACTTGTGAGCAGCTACACTTCCATGTGCTGGATACATCAGATTACCTCCGGCCAGTG
2221 + + + + + + 2280
CAGTGAACACTCGTCGATGTGAAGGTACACGACCTATGTAGTCTAATGGAGGCCGGTCAC
V T C E Q L H F H V L D T S D Y L R P V
GCCTTGACTGTGACCTTTGCCTTGGACAATACTACAAAGCCAGGGCCTGTGCTGAATGAG
CGGAACTGACACTGGAAACGGAACCTGTTATGATGTTTCGGTCCCGGACACGACTTACTC
A L T V T F A L D N T T K P G P V L N E
GGCTCACCCACCTCTATACAAAAGCTGGTCCCCTTCTCAAAGGATTGTGGCCCTGACAAT
2341 + + + + + + 2400
CCGAGTGGGTGGAGATATGTTTTCGACCAGGGGAAGAGTTTCCTAACACCGGGACTGTTA
G S P T S I Q K L V P F S K D C G P D N 40
GAATGTGTCACAGACCTGGTGCTTCAAGTGAATATGGACATCAGAGGCTCCAGGAAGGCC
2401 + + + + + + 2460
CTTACACAGTGTCTGGACCACGAAGTTCACTTATACCTGTAGTCTCCGAGGTCCTTCCGG
E C V T D V L Q V N M D I R G S R K A
CCATTTGTGGTTCGAGGTGGCCGGCGGAAAGTGCTGGTATCTACAACTCTGGAGAACAGA
GGTAAACACCAAGCTCCACCGGCCGCCTTTCACGACCATAGATGTTGAGACCTCTTGTCT
P F V V R G G R R K V L V S T T E N R
AAGGAAAATGCTTACAATACGAGCCTGAGTATCATCTTCTCTAGAAACCTCCACCTGGCC
2521 + + + + + + 2580
TTCCTTTTACGAATGTTATGCTCGGACTCATAGTAGAAGAGATCTTTGGAGGTGGACCGG
K E N A Y N T S L S I I F S R N L H L A
AGTCTCACTCCTCAGAGAGAGAGCCCAATAAAGGTGGAATGTGCCGCCCCTTCTGCTCAT
TCAGAGTGAGGAGTCTCTCTCTCGGGTTATTTCCACCTTACACGGCGGGGAAGACGAGTA
S L T P Q R E S P I K V E C A A P S A H
GCCCGGCTCTGCAGTGTGGGGCATCCTGTCTTCCAGACTGGAGCCAAGGTGACCTTTCTG
CGGGCCGAGACGTCACACCCCGTAGGACAGAAGGTCTGACCTCGGA'CCACTGGAAAGAC
A R L C S V G H P V F Q T G A K V T F L
CTAGAGTTTGAGTTTAGCTGCTCCTCTCTCCTGAGCCAGGTCTTTGGGAAGCTGACTGCC
GATCTCAAACTCAAATCGACGAGGAGAGAGGACTCGGTCCAGAAACCCTTCGACTGACGG
L E F E F S C S S L L S Q V F G K L T A
AGCAGTGACAGCCTGGAGAGAAATGGCACCCTTCAAGAAAACACAGCCCAGACCTCAGCC
2761 + + + + + + 2820
TCGTCACTGTCGGACCTCTCTTTACCGTGGGAAGTTCTTTTGTGTCGGGTCTGGAGTCGG
S S D S L E R N G T Q E N T A Q T S A
TACATCCAATATGAGCCCCACCTCCTGTTCTCTAGTGAGTCTACCCTGCACCGCTATGAG
2821 + + + + + + 2880
ATGTAGGTTATACTCGGGGTGGAGGACAAGAGATCACTCAGATGGGACGTGGCGATACTC
Y I Q Y E P H L L F S S E S T H R Y E
GTTCACCCATATGGGACCCTCCCAGTGGGTCCTGGCCCAGAATTCAAAACCACTCTCAGG
2881 + + + + + + 2940
CAAGTGGGTATACCCTGGGAGGGTCACCCAGGACCGGGTCTTAAGTTTTGGTGAGAGTCC
V H P Y G T L P V G P G P E F K T T L R
GTTCAGAACCTAGGCTGCTATGTGGTCAGTGGCCTCATCATCTCAGCCCTCCTTCCAGCT
2941 + + + + + + 3000
CAAGTCTTGGATCCGACGATACACCAGTCACCGGAGTAGTAGAGTCGGGAGGAAGGTCGA
V Q N L G C Y V V S G I I S A L P A
GTGGCCCATGGGGGCAATTACTTCCTATCACTGTCTCAAGTCATCACTAACAATGCAAGC
3001 + + + + + + 3060
CACCGGGTACCCCCGTTAATGAAGGATAGTGACAGAGTTCAGTAGTGATTGTTACGTTCG
V A H G G N Y F S L S Q V I T N N A S 41
TGCATAGTGCAGAACCTGACTGAACCCCCAGGCCCACCTGTGCATCCAGAGGAGCTTCAA
3061 + + + + + + 3120
ACGTATCACGTCTTGGACTGACTTGGGGGTCCGGGTGGACACGTAGGTCTCCTCGAAGTT
C I V Q N L T E P P G P P V H P E E L Q
CACACAAACAGACTGAATGGGAGCAATACTCAGTGTCAGGTGGTGAGGTGCCACCTTGGG
3121 + + + + + + 3180
GTGTGTTTGTCTGACTTACCCTCGTTATGAGTCACAGTCCACCACTCCACGGTGGAACCC
H T N R L N G S N T Q C Q V V R C H L G
CAGCTGGCAAAGGGGACTGAGGTCTCTGTTGGACTATTGAGGCTGGTTCACAATGAATTT
GTCGACCGTTTCCCCTGACTCCAGAGACAACCTGATAACTCCGACCAAGTGTTACTTAAA
Q L A K G T E V S V G L L R L V H N E F
TTCCGAAGAGCCAAGTTCAAGTCCCTGACGGTGGTCAGCACCTTTGAGCTGGGAACCGAA
AAGGCTTCTCGGTTCAAGTTCAGGGACTGCCACCAGTCGTGGAAACTCGACCCTTGGCTT
F R R A K F K S L T V V S T F E L G T E
GAGGGCAGTGTCCTACAGCTGACTGAAGCCTCCCGTTGGAGTGAGAGCCTCTTGGAGGTG
CTCCCGTCACAGGATGTCGACTGACTTCGGAGGGCAACCTCACTCcGGAGAACCTCCAC
E G S V L Q L T E A S R S E S L L E V
GTTCAGACCCGGCCTATCCTCATCTCCCTGTGGATCCTCATAGGCAGTGTCCTGGGAGGG
3361 + + + + + + 3420
CAAGTCTGGGCCGGATAGGAGTAGAGGGACACCTAGGAGTATCCGTCACAGGACCCTCCC
V Q T R P I L I S L I L I G S V L G G
TTGCTCCTGCTTGCTCTCCTTGTCTTCTGCCTGTGGAAGCTTGGCTTCTTTGCCCATAAG
AACGAGGACGAACGAGAGGAACAGAAGACGGACACCTTCGAACCGAAGAAACGGGTATTC
L L L L A L L V F C L K L G F F A H K
AAAATCCCTGAGGAAGAAAAAAGAGAAGAGAAGTTGGAGCAATGAATGTAGAATAAGGGT
3481 + + + + + + 3540
TTTTAGGGACTCCTTCTTTTTTCTCTTCTCTTCAACCTCGTTACTTACATCTTATTCCCA
K I P E E E K R E E K L E Q
CTAGAAAGTCCTCCCTGGCAGCTTTCTTCAAGAGACTTGCATAAAAGCAGAGGTTTGGGG
3541 + + + + + + 3600
GATCTTTCAGGAGGGACCGTCGAAAGAAGTTCTCTGAACGTATTTTCGTCTCCAAACCCC
GCTCAGATGGGACAAGAAGCCGCCTCTGGACTATCTCCCCAGACCAGCAGCCTGACTTGA CGAGTCTACCCTGTTCTTCGGCGGAGACCTGATAGAGGGGTCTGGTCGTCGGACTGAACT
CTTTTGAGTCCTAGGGATGCTGCTGGCTAGAGATGAGGCTTTACCTCAGACAAGAAGAGC GAAAACTCAGGATCCCTACGACGACCGATCTCTACTCCGAAATGGAGTCTGTTCTTCTCG 42
TGGCACCAAAACTAGCCATGCTCCCACCCTCTGCTTCCCTCCTCCTCGTGATCCTGGTTC ACCGTGGTTTTGATCGGTACGAGGGTGGGAGACGAAGGGAGGAGGAGCACTAGGACCAAG
CATAGCCAACACTGGGGCTTTTGTTTGGGGTCCTTTTATCCCCAGGAATCAATAATTTTT
3781 + + + + + + 3840
GTATCGGTTGTGACCCCGAAAACAAACCCCAGGAAAATAGGGGTCCTTAGTTATTAAAAA
TTGCCTAGGAAAAAAAAAAGCGGCCGCGAATTCGATATCAAGCT
3841 + + + + 3884
AACGGATCCTTTTTTTTTTCGCCGGCGCTTAAGCTATAGTTCGA
43
(2) INFORMATION FOR SEQ ID NO. 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3779 base pairs
(B) TYPE: nucleid acid and amino acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (E)
(i) MOLECULAR TYPE: cDNA (vi) ORIGINAL SOURCE:
(A) ORGANISM: human
(B) CELLTYPE: chondrocyte
(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 2:
CAGGTCAGAAACCGATCAGGCATGGAACTCCCCTTCGTCACTCACCTGTTCTTGCCCCTG GTCCAGTCTTTGGCTAGTCCGTACCTTGAGGGGAAGCAGTGAGTGGACAAGAACGGGGAC
M E P F V T H L F L P GTGTTCCTGACAGGTCTCTGCTCCCCCTTTAACCTGGATGAACATCACCCACGCCTATTC CACAAGGACTGTCCAGAGACGAGGGGGAAATTGGACCTACTTGTAGTGGGTGCGGATAAG V F L T G L C S P F N D E H H P R L F CCAGGGCCACCAGAAGCTGAATTTGGATACAGTGTCTTACAACATGTTGGGGGTGGACAG GGTCCCGGTGGTCTTCGACTTAAACCTATGTCACAGAATGTTGTACAACCCCCACCTGTC P G P P E A E F G Y S V Q H V G G G Q CGATGGATGCTGGTGGGCGCCCCCTGGGATGGGCCTTCAGGCGACCGGAGGGGGGACGTT GCTACCTACGACCACCCGCGGGGGACCCTACCCGGAAGTCCGCTGGCCTCCCCCCTGCAA R M L V G A P D G P S G D R R G D V TATCGCTGCCCTGTAGGGGGGGCCCACAATGCCCCATGTGCCAAGGGCCACTTAGGTGAC ATAGCGACGGGACATCCCCCCCGGGTGTTACGGGGTACACGGTTCCCGGTGAATCCACTG Y R C P V G G A H N A P C A K G H L G D TACCAACTGGGAAATTCATCTCATCCTGCTGTGAATATGCACCTGGGGATGTCTCTGTTA ATGGTTGACCCTTTAAGTAGAGTAGGACGACACTTATACGTGGACCCCTACAGAGACAAT Y Q L G N S S H P A V N M H L G M S L L GAGACAGATGGTGATGGGGGATTCATGGCCTGTGCCCCTCTCTGGTCTCGTGCTTGTGGC CTCTGTCTACCACTACCCCCTAAGTACCGGACACGGGGAGAGACCAGAGCACGAACACCG E T D G D G G F M A C A P L W S R A C G AGCTCTGTCTTCAGTTCTGGGATATGTGCCCGTGTGGATGCTTCATTCCAGCCTCAGGGA TCGAGACAGAAGTCAAGACCCTATACACGGGCACACCTACGAAGTAAGGTCGGAGTCCCT 44 a S S V F S S G I C A R V D A S F Q P Q G
AGCCTGGCACCCACTGCCCAACGCTGCCCAACATACATGGATGTTGTCATTGTCTTGGAT
481 + + + + + + 540
TCGGACCGTGGGTGACGGGTTGCGACGGGTTGTATGTACCTACAACAGTAACAGAACCTA a S A P T A Q R C P T Y M D V V I V D
GGCTCCAACAGCATCTACCCCTGGTCTGAAGTTCAGACCTTCCTACGAAGACTGGTAGGG
541 + + + + + + 600
CCGAGGTTGTCGTAGATGGGGACCAGACTTCAAGTCTGGAAGGATGCTTCTGACCATCCC a G S N S I Y P S E V Q T F R R L V G
AAACTGTTTATTGACCCAGAACAGATACAGGTGGGACTGGTACAGTATGGGGAGAGCCCT
601 + + + + + + 660
TTTGACAAATAACTGGGTCTTGTCTATGTCCACCCTGACCATGTCATACCCCTCTCGGGA a K L F I D P E Q I Q V G L V Q Y G E S P
GTACATGAGTGGTCCCTGGGAGATTTCCGAACGAAGGAAGAAGTGGTGAGAGCAGCAAAG CATGTACTCACCAGGGACCCTCTAAAGGCTTGCTTCCTTCTTCACCACTCTCGTCGTTTC a V H E W S L G D F R T K E E V V R A A K
AACCTCAGTCGGCGGGAGGGACGAGAAACAAAGACTGCCCAAGCAATAATGGTGGCCTGC TTGGAGTCAGCCGCCCTCCCTGCTCTTTGTTTCTGACGGGTTCGTTATTACCACCGGACG a N L S R R E G R E T K T A Q A I M V A C
ACAGAAGGGTTCAGTCAGTCCCATGGGGGCCGACCCGAGGCTGCCAGGCTACTGGTGGTT TGTCTTCCCAAGTCAGTCAGGGTACCCCCGGCTGGGCTCCGACGGTCCGATGACCACCAA a T E G F S Q S H G G R P E A A R L V V
GTCACTGATGGAGAGTCCCATGATGGAGAGGAGCTTCCTGCAGCACTAAAGGCCTGTGAG CAGTGACTACCTCTCAGGGTACTACCTCTCCTCGAAGGACGTCGTGATTTCCGGACACTC a V T D G E S H D G E E L P A A K A C E
GCTGGAAGAGTGACACGCTATGGGATTGCAGTCCTTGGTCACTACCTCCGGCGGCAGCGA
901 + + + + + + 960
CGACCTTCTCACTGTGCGATACCCTAACGTCAGGAACCAGTGATGGAGGCCGCCGTCGCT a A G R V T R Y G I A V L G H Y L R R Q R
GATCCCAGCTCTTTCCTGAGAGAAATTAGAACTATTGCCAGTGATCCAGATGAGCGATTC
961 + + + + + + 1020
CTAGGGTCGAGAAAGGACTCTCTTTAATCTTGATAACGGTCACTAGGTCTACTCGCTAAG a D P S S F L R E I R T I A S D P D E R F
TTCTTCAATGTCACAGATGAGGCTGCTCTGACTGACATTGTGGATGCACTAGGAGATCGG
1021 + + + + + + 1080
AAGAAGTTACAGTGTCTACTCCGACGAGACTGACTGTAACACCTACGTGATCCTCTAGCC a F F N V T D E A A L T D I V D A L G D R
ATTTTTGGCCTTGAAGGGTCCCATGCAGAAAACGAAAGCTCCTTTGGGCTGGAAATGTCT
1081 + + + + + + 1140
TAAAAACCGGAACTTCCCAGGGTACGTCTTTTGCTTTCGAGGAAACCCGACCTTTACAGA 45 a I F G L E G S H A E N E S S F G L E M S
CAGATTGGTTTCTCCACTCATCGGCTAAAGGATGGGATTCTTTTTGGGATGGTGGGGGCC GTCTAACCAAAGAGGTGAGTAGCCGATTTCCTACCCTAAGAAAAACCCTACCACCCCCGG a Q I G F S T H R L K D G I L F G M V G A
TATGACTGGGGAGGCTCTGTGCTATGGCTTGAAGGAGGCCACCGCCTTTTCCCCCCACGA
1201 + + + + + + 1260
ATACTGACCCCTCCGAGACACGATACCGAACTTCCTCCGGTGGCGGAAAAGGGGGGTGCT a Y D W G G S V L L E G G H R L F P P R
ATGGCACTGGAAGACGAGTTCCCCCCTGCACTGCAGAACCATGCAGCCTACCTGGGTTAC TACCGTGACCTTCTGCTCAAGGGGGGACGTGACGTCTTGGTACGTCGGATGGACCCAATG a M A L E D E F P P A L Q N H A A Y L G Y
TCTGTTTCTTCCATGCTTTTGCGGGGTGGACGCCGCCTGTTTCTCTCTGGGGCTCCTCGA AGACAAAGAAGGTACGAAAACGCCCCACCTGCGGCGGACAAAGAGAGACCCCGAGGAGCT a S V S S M L L R G G R R L F L S G A P R
TTTAGACATCGAGGAAAAGTCATCGCCTTCCAGCTTAAGAAAGATGGGGCTGTGAGGGTT
1381 + + + + - + + 1440
AAATCTGTAGCTCCTTTTCAGTAGCGGAAGGTCGAATTCTTTCTACCCCGACACTCCCAA a F R H R G K V I A F Q L K K D G A V R V
GCCCAGAGCCTCCAGGGGGAGCAGATTGGTTCATACTTTGGCAGTGAGCTCTGCCCATTG
1441 + + + + + + 1500
CGGGTCTCGGAGGTCCCCCTCGTCTAACCAAGTATGAAACCGTCACTCGAGACGGGTAAC a A Q S L Q G E Q I G S Y F G S E L C P L
GATACAGATAGGGATGGAACAACTGATGTCTTACTTGTGGCTGCCCCCATGTTCCTGGGA
1501 + + + + + + 1560
CTATGTCTATCCCTACCTTGTTGACTACAGAATGAACACCGACGGGGGTACAAGGACCCT a D T D R D G T T D V L L V A A P M F L G
CCCCAGAACAAGGAAACAGGACGTGTTTATGTGTATCTGGTAGGCCAGCAGTCCTTGCTG
1561 + + + + + + 1620
GGGGTCTTGTTCCTTTGTCCTGCACAAATACACATAGACCATCCGGTCGTCAGGAACGAC a P Q N K E T G R V Y V Y L V G Q Q S L L
ACCCTCCAAGGAACACTTCAGCCAGAACCCCCCCAGGATGCTCGGTTTGGCTTTGCCATG TGGGAGGTTCCTTGTGAAGTCGGTCTTGGGGGGGTCCTACGAGCCAAACCGAAACGGTAC a T Q G T L Q P E P P Q D A R F G F A M
GGAGCTCTTCCTGATCTGAACCAAGATGGTTTTGCTGATGTGGCTGTGGGGGCGCCTCTG
1681 + + + + + + 1740
CCTCGAGAAGGACTAGACTTGGTTCTACCAAAACGACTACACCGACACCCCCGCGGAGAC a G A L P D L N Q D G F A D V A V G A P L
GAAGATGGGCACCAGGGAGCACTGTACCTGTACCATGGAACCCAGAGTGGAGTCAGGCCC
1741 + + + + + + 1800
CTTCTACCCGTGGTCCCTCGTGACATGGACATGGTACCTTGGGTCTCACCTCAGTCCGGG 46 a E D G H Q G A L Y L Y H G T Q S G V R P
CATCCTGCCCAGAGGATTGCTGCTGCCTCCATGCCACATGCCCTCAGCTACTTTGGCCGA
1801 + + + + + + 1860
GTAGGACGGGTCTCCTAACGACGACGGAGGTACGGTGTACGGGAGTCGATGAAACCGGCT a H P A Q R I A A A S M P H A L S Y F G R
AGTGTGGATGGTCGGCTAGATCTGGATGGAGATGATCTGGTCGATGTGGCTGTGGGTGCC
1861 + + + + + + 1920
TCACACCTACCAGCCGATCTAGACCTACCTCTACTAGACCAGCTACACCGACACCCACGG a S V D G R L D L D G D D L V D V A V G A
CAGGGGGCAGCCATCCTGCTCAGCTCCCGGCCCATTGTCCATCTGACCCCATCACTGGAG
1921 + + + + + + 1980
GTCCCCCGTCGGTAGGACGAGTCGAGGGCCGGGTAACAGGTAGACTGGGGTAGTGACCTC a Q G A A I L S S R P I V H T P S L E
GTGACCCCACAGGCCATCAGTGTGGTTCAGAGGGACTGTAGGCGGCGAGGCCAAGAAGCA
1981 + + + + + + 2040
CACTGGGGTGTCCGGTAGTCACACCAAGTCTCCCTGACATCCGCCGCTCCGGTTCTTCGT a V T P Q A I S V V Q R D C R R R G Q E A
GTCTGTCTGACTGCAGCCCTTTGCTTCCAAGTGACCTCCCGTACTCCTGGTCGCTGGGAT
2041 + + + + 1 + + 2100
CAGACAGACTGACGTCGGGAAACGAAGGTTCACTGGAGGGCATGAGGACCAGCGACCCTA a V C T A A L C F Q V T S R T P G R D
CACCAATTCTACATGAGGTTCACCGCATCACTGGATGAATGGACTGCTGGGGCACGTGCA
2101 + + + + + + 2160
GTGGTTAAGATGTACTCCAAGTGGCGTAGTGACCTACTTACCTGACGACCCCGTGCACGT a H Q F Y M R F T A S L D E W T A G A R A
GCATTTGATGGCTCTGGCCAGAGGTTGTCCCCTCGGAGGCTCCGGCTCAGTGTGGGGAAT
2161 + + + + + + 2220
CGTAAACTACCGAGACCGGTCTCCAACAGGGGAGCCTCCGAGGCCGAGTCACACCCCTTA a A F D G S G Q R L S P R R L R L S V G N
GTCACTTGTGAGCAGCTACACTTCCATGTGCTGGATACATCAGATTACCTCCGGCCAGTG
2221 + + + + + + 2280
CAGTGAACACTCGTCGATGTGAAGGTACACGACCTATGTAGTCTAATGGAGGCCGGTCAC a V T C E Q L H F H V L D T S D Y L R P V
GCCTTGACTGTGACCTTTGCCTTGGACAATACTACAAAGCCAGGGCCTGTGCTGAATGAG
2281 + + + + + + 2340
CGGAACTGACACTGGAAACGGAACCTGTTATGATGTTTCGGTCCCGGACACGACTTACTC a A L T V T F A D N T T K P G P V L N E
GGCTCACCCACCTCTATACAAAAGCTGGTCCCCTTCTCAAAGGATTGTGGCCCTGACAAT
2341 + + + + + + 2400
CCGAGTGGGTGGAGATATGTTTTCGACCAGGGGAAGAGTTTCCTAACACCGGGACTGTTA a G S P T S I Q K L V P F S K D C G P D N
GAATGTGTCACAGACCTGGTGCTTCAAGTGAATATGGACATCAGAGGCTCCAGGAAGGCC
2401 + + + + + + 2460
CTTACACAGTGTCTGGACCACGAAGTTCACTTATACCTGTAGTCTCCGAGGTCCTTCCGG 47 a E C V T D V L Q V N M D I R G S R K A
CCATTTGTGGTTCGAGGTGGCCGGCGGAAAGTGCTGGTATCTACAACTCTGGAGAACAGA
2461 + + + + + + 2520
GGTAAACACCAAGCTCCACCGGCCGCCTTTCACGACCATAGATGTTGAGACCTCTTGTCT a P F V V R G G R R K V L V S T T E N R
AAGGAAAATGCTTACAATACGAGCCTGAGTATCATCTTCTCTAGAAACCTCCACCTGGCC
2521 + + + + + + 2580
TTCCTTTTACGAATGTTATGCTCGGACTCATAGTAGAAGAGATCTTTGGAGGTGGACCGG a K E N A Y N T S L S I I F S R N H L A
AGTCTCACTCCTCAGAGAGAGAGCCCAATAAAGGTGGAATGTGCCGCCCCTTCTGCTCAT
2581 + + + + + + 2640
TCAGAGTGAGGAGTCTCTCTCTCGGGTTATTTCCACCTTACACGGCGGGGAAGACGAGTA a S L T P Q R E S P I K V E C A A P S A H
GCCCGGCTCTGCAGTGTGGGGCATCCTGTCTTCCAGACTGGAGCCAAGGTGACCTTTCTG CGGGCCGAGACGTCACACCCCGTAGGACAGAAGGTCTGACCTCGGTTCCACTGGAAAGAC a A R L C S V G H P V F Q T G A K V T F L
CTAGAGTTTGAGTTTAGCTGCTCCTCTCTCCTGAGCCAGGTCTTTGGGAAGCTGACTGCC
2701 + + + + + + 2760
GATCTCAAACTCAAATCGACGAGGAGAGAGGACTCGGTCCAGAAACCCTTCGACTGACGG a L E F E F S C S S L S Q V F G K L T A
AGCAGTGACAGCCTGGAGAGAAATGGCACCCTTCAAGAAAACACAGCCCAGACCTCAGCC
2761 + + + + + + 2820
TCGTCACTGTCGGACCTCTCTTTACCGTGGGAAGTTCTTTTGTGTCGGGTCTGGAGTCGG a S S D S E R N G T L Q E N T A Q T S A
TACATCCAATATGAGCCCCACCTCCTGTTCTCTAGTGAGTCTACCCTGCACCGCTATGAG
2821 + + + + + + 2880
ATGTAGGTTATACTCGGGGTGGAGGACAAGAGATCACTCAGATGGGACGTGGCGATACTC a Y I Q Y E P H L L F S S E S T L H R Y E
GTTCACCCATATGGGACCCTCCCAGTGGGTCCTGGCCCAGAATTCAAAACCACTCTCAGG
2881 + + + + + + 2940
CAAGTGGGTATACCCTGGGAGGGTCACCCAGGACCGGGTCTTAAGTTTTGGTGAGAGTCC a V H P Y G T L P V G P G P E F K T T L R
ACTAACAATGCAAGCTGCATAGTGCAGAACCTGACTGAACCCCCAGGCCCACCTGTGCAT TGATTGTTACGTTCGACGTATCACGTCTTGGACTGACTTGGGGGTCCGGGTGGACACGTA a T N N A S C I V Q N L T E P P G P P V H
CCAGAGGAGCTTCAACACACAAACAGACTGAATGGGAGCAATACTCAGTGTCAGGTGGTG
3001 + + + + + + 3060
GGTCTCCTCGAAGTTGTGTGTTTGTCTGACTTACCCTCGTTATGAGTCACAGTCCACCAC a P E E L Q H T N R L N G S N T Q C Q V V
AGGTGCCACCTTGGGCAGCTGGCAAAGGGGACTGAGGTCTCTGTTGGACTATTGAGGCTG
TCCACGGTGGAACCCGTCGACCGTTTCCCCTGACTCCAGAGACAACCTGATAACTCCGAC 48 a R C H L G Q L A K G T E V S V G L L R L
GTTCACAATGAATTTTTCCGAAGAGCCAAGTTCAAGTCCCTGACGGTGGTCAGCACCTTT
3121 + + + + + + 3180
CAAGTGTTACTTAAAAAGGCTTCTCGGTTCAAGTTCAGGGACTGCCACCAGTCGTGGAAA a V H N E F F R R A K F K S L T V V S T F
GAGCTGGGAACCGAAGAGGGCAGTGTCCTACAGCTGACTGAAGCCTCCCGTTGGAGTGAG
3181 + + + + + + 3240
CTCGACCCTTGGCTTCTCCCGTCACAGGATGTCGACTGACTTCGGAGGGCAACCTCACTC a E L G T E E G S V L Q L T E A S R W S E
AGCCTCTTGGAGGTGGTTCAGACCCGGCCTATCCTCATCTCCCTGTGGATCCTCATAGGC
3241 + + + + + + 3300
TCGGAGAACCTCCACCAAGTCTGGGCCGGATAGGAGTAGAGGGACACCTAGGAGTATCCG a S L E V V Q T R P I L I S L I L I G
AGTGTCCTGGGAGGGTTGCTCCTGCTTGCTCTCCTTGTCTTCTGCCTGTGGAAGCTTGGC
3301 + + + + + + 3360
TCACAGGACCCTCCCAACGAGGACGAACGAGAGGAACAGAAGACGGACACCTTCGAACCG a S V L G G L L L L A L V F C L K L G
TTCTTTGCCCATAAGAAAATCCCTGAGGAAGAAAAAAGAGAAGAGAAGTTGGAGCAATGA AAGAAACGGGTATTCTTTTAGGGACTCCTTCTTTTTTCTCTTCTCTTCAACCTCGTTACT a F F A H K K I P E E E K R E E K L E Q
ATGTAGAATAAGGGTCTAGAAAGTCCTCCCTGGCAGCTTTCTTCAAGAGACTTGCATAAA
3421 + + + + + + 3480
TACATCTTATTCCCAGATCTTTCAGGAGGGACCGTCGAAAGAAGTTCTCTGAACGTATTT
AGCAGAGGTTTGGGGGCTCAGATGGGACAAGAAGCCGCCTCTGGACTATCTCCCCAGACC
3481 + + + + + + 3540
TCGTCTCCAAACCCCCGAGTCTACCCTGTTCTTCGGCGGAGACCTGATAGAGGGGTCTGG
AGCAGCCTGACTTGACTTTTGAGTCCTAGGGATGCTGCTGGCTAGAGATGAGGCTTTACC
3541 + + + + + + 3600
TCGTCGGACTGAACTGAAAACTCAGGATCCCTACGACGACCGATCTCTACTCCGAAATGG
TCAGACAAGAAGAGCTGGCACCAAAACTAGCCATGCTCCCACCCTCTGCTTCCCTCCTCC AGTCTGTTCTTCTCGACCGTGGTTTTGATCGGTACGAGGGTGGGAGACGAAGGGAGGAGG
TCGTGATCCTGGTTCCATAGCCAACACTGGGGCTTTTGTTTGGGGTCCTTTTATCCCCAG AGCACTAGGACCAAGGTATCGGTTGTGACCCCGAAAACAAACCCCAGGAAAATAGGGGTC
GAATCAATAATTTTTTTGCCTAGGAAAAAAAAAAGCGGCCGCGAATTCGATATCAAGCT
3721 + + + + + 3779
CTTAGTTATTAAAAAAACGGATCCTTTTTTTTTTCGCCGGCGCTTAAGCTATAGTTCGA 49 (2) INFORMATION FOR SEQ ID NO. 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 143 base pairs
(B) TYPE: nucleic acid and amino acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(iii) MOLECULAR TYPE: cDNA (vi) ORIGINAL SOURCE:
(A) ORGANISM: human
(B) CELLTYPE: chondrocyte
(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 3:
Ndel
I GGGGCATATGGTTCAGAACCTGGGTTGCTACGTTGTTTCCGGTCTGATCATCTCCGCTCT
CCCCGTATACCAAGTCTTGGACCCAACGATGCAACAAAGGCCAGACTAGTAGAGGCGAGA
G H M V Q N L G C Y V V S G L. il I S A L -
GCTGCCGGCTGTTGCTCACGGTGGTAACTACTTCCTAAGCTTGTCCCAGGTTATCAGCGG 61 + + + + + + 120
CGACGGCCGACAACGAGTGCCACCATTGATGAAGGATTCGAACAGGGTCCAATAGTCGCC
L P A V A H G G N Y F L S L S Q V I S G -
BamHI I CCTGGTGCCGCGCGGATCCCCCC 121 + +— 143
GGACCACGGCGCGCCTAGGGGGG
L V P R G S P

Claims

50CLAIMS
1. A recombinant or isolated integrin subunit ╬▒lO comprising the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, or homologues or fragments thereof having similar biological activity.
2. A process of producing a recombinant integrin subunit ╬▒lO comprising the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, or homologues or fragments thereof having similar biological activity, which process comprises the steps of a) isolating a polynucleotide comprising a nucleotide sequence coding for an integrin subunit ╬▒lO, or homologues or fragments thereof having similar biological activity, b) constructing an expression vector comprising the isolated polynucleotide, c) transforming a host cell with said expression vector, d) culturing said transformed host cell in a culture medium under conditions suitable for expression of integrin subunit ╬▒lO, or homologues or fragments thereof having similar biological activity, in said transformed host cell, and, optionally, e) isolating the integrin subunit ╬▒lO, or homologues or fragments thereof having similar biological activity, from said transformed host cell or said culture medium.
3. A process of providing an integrin subunit ╬▒lO, or homologues or fragments thereof having similar biological activity, whereby said subunit is isolated from a cell in which it is naturally present.
4. An isolated polynucleotide comprising a nucleotide coding for an integrin subunit ╬▒lO, or for homolo- gues or fragments thereof, which polynucleotide comprises the nucleotide sequence shown in SEQ ID No. 1 or SEQ ID No. 2 or suitable parts thereof. 51
5. An isolated polynucleotide or oligonucleotide which hybridises to a DNA or RNA encoding an integrin subunit ╬▒lO or homologues or fragments thereof, wherein said polynucleotide or oligonucleotide fails to hybridise to a DNA or RNA encoding an integrin subunit ╬▒l.
6. A vector comprising a polynucleotide or oligonucleotide coding for an integrin subunit ╬▒lO, or homologues or fragments thereof, which polynucleotide or oli- gonucleotide comprises the nucleotide sequence shown in SEQ ID No. 1 or SEQ ID No. 2 or parts thereof.
7. A vector comprising a polynucleotide or oligonucleotide which hybridises to a DNA or RNA encoding an integrin subunit ╬▒lO or homologues or fragments thereof, wherein said polynucleotide or oligonucleotide fails to hybridise to a DNA or RNA encoding an integrin subunit ╬▒l.
8. A cell containing the vector as defined in any one of claims 6 and 7.
9. A cell generated by the process in claim 2, in which a polynucleotide or oligonucleotide coding for an integrin subunit ╬▒lO, or homologues or fragments thereof, which polynucleotide or oligonucleotide comprises the nucleotide sequence shown in SEQ ID No. 1 or SEQ ID No. 2 or parts thereof has been stably integrated in the cell genome .
10. Binding entities having the capability of binding specifically to integrin subunit ╬▒lO comprising the amino acid sequence of SEQ ID No. 1 or SEQ ID No. 2, or to homologues or fragments thereof.
11. Binding entities according to claim 10, which are chosen from the group comprising proteins, peptides, carbohydrates, lipids, natural integrin binding ligands, polyclonal and monoclonal antibodies, and fragments thereof.
12. A recombinant or isolated integrin heterodimer comprising a subunit ╬▒lO and a subunit ╬▓, in which the 52 subunit ╬▒lO comprises the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, and homologues and fragments thereof having similar biological activity.
13. A recombinant or isolated integrin heterodimer according to claim 12, wherein the subunit ╬▓ is ╬▓l.
14. A process of producing a recombinant integrin heterodimer comprising a subunit ╬▒lO and a subunit ╬▓, in which the subunit ╬▒lO comprises the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, and homologues and fragments thereof, which process comprises the steps of a) isolating one polynucleotide comprising a nucleotide sequence coding for a subunit ╬▒lO of an integrin heterodimer and, optionally, another polynucleotide comprising a nucleotide sequence coding for a subunit ╬▓ of an integrin heterodimer, or polynucleotides or oligo- nucleotides coding for homologues or fragments thereof having similar biological activity, b) constructing an expression vector comprising said isolated polynucleotide coding for said subunit ╬▒lO optionally in combination with an expression vector comprising said isolated nucleotide coding for said subunit ╬▓, c) transforming a host cell with said expression vector or vectors, d) culturing said transformed host cell in a culture medium under conditions suitable for expression of an integrin heterodimer comprising a subunit ╬▒lO and a subunit ╬▓, or homologues or fragments thereof having similar biological activity, in said transformed host cell, and, optionally, e) isolating the integrin heterodimer comprising a subunit ╬▒lO and a subunit ╬▓, or homologues or fragments thereof having similar biological activity, or the ╬▒lO subunit thereof from said transformed host cell or said culture medium.
15. A process of providing an integrin heterodimer comprising a subunit ╬▒lO and a subunit ╬▓, or homologues 53 or fragments thereof having similar biological activity, whereby said integrin heterodimer is isolated from a cell in which it is naturally present.
16. A cell containing a first vector, said first vector comprising a polynucleotide or oligonucleotide coding for a subunit ╬▒lO of an integrin heterodimer, or for homologues or parts thereof having similar biological activity, which polynucleotide or oligonucleotide comprises the nucleotide sequence shown in SEQ ID No. 1 or SEQ ID No. 2 or parts thereof, and a second vector, said second vector comprising a polynucleotide or oligonucleotide coding for a subunit ╬▓ of an integrin heterodimer, or for homologues or fragments thereof.
17. Binding entities having the capability of bind- ing specifically to the integrin heterodimer comprising a subunit ╬▒lO and a subunit ╬▓, or to homologues or fragments thereof, or a subunit ╬▒lO thereof, having similar biological activity.
18. Binding entities according to claim 17, wherein the subunit ╬▓ is ╬▓l.
19. Binding entities according to claim 17 or 18, which are chosen among the group comprising proteins, peptides, carbohydrates, lipids, natural integrin binding ligands, and fragments thereof.
20. A fragment of the integrin subunit ╬▒lO, which fragment is a peptide chosen from the group comprising peptides of the cytoplasmic domain, the I-domain and the spliced domain.
21. A fragment according to claim 20, which is a peptide comprising the amino acid sequence
KLGFFAHKKIPEEEKREEKLEQ.
22. A fragment according to claim 20, which comprises the amino acid sequence from about amino acid No. 952 to about amino acid no. 986 of SEQ ID No. 1.
23. A fragment according to claim 20, which is a peptide comprising the amino acid sequence from about 54 amino acid No. 140 to about amino acid no. 337 of SEQ ID No. 1.
24. A method of producing a fragment of the integrin subunit ╬▒lO as defined in any one of claims 20-23, which method comprises a sequential addition of amino acids containing protective groups.
25. A polynucleotide or oligonucleotide coding for a fragment of the integrin subunit ╬▒lO as defined in any one of claims 20-23.
26. Binding entities having the capability of binding specifically to a fragment of the human integrin subunit ╬▒lO as defined in any one of claims 20-23.
27. Binding entities according to claim 26, which are chosen from the group comprising proteins, peptides, carbohydrates, lipids, natural integrin binding ligands, and fragments thereof.
28. A process of using an integrin subunit ╬▒lO comprising the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, or an integrin heterodimer comprising said subunit ╬▒lO and a subunit ╬▓, or a homologue or fragment of said integrin or subunit having similar biological activity, as a marker or target molecule of cells or tissues expressing said integrin subunit ╬▒lO, which cells or tissues are of animal including human origin.
29. A process according to claim 28, whereby said fragment is a peptide chosen from the group comprising peptides of the cytoplasmic domain, the I-domain and the spliced domain.
30. A process according to claim 29, whereby said fragment is a peptide comprising the amino acid sequence
KLGFFAHKKIPEEEKREEKLEQ.
31. A process according to claim 29, whereby said fragment comprises the amino acid sequence from about amino acid no. 952 to about amino acid no. 986 of SEQ ID No. 1.
32. A process according to claim 29, whereby said fragment comprises the amino acid sequence from about 55 amino acid no. 140 to about amino acid no. 337 of SEQ ID No. 1.
33. A process according to claim 28, whereby the subunit ╬▓ is ╬▓l.
34. A process according to claim 28, whereby said cells are chosen from the group comprising chondrocytes, smooth muscle cells, endothelial cells, osteoblasts and fibroblasts.
35. A process according to any one of claims 28-34, which process is used during pathological conditions involving said subunit ╬▒lO.
36. A process according to claim 35, which pathological conditions comprise damage of cartilage.
37. A process according to claim 36, which patho- logical conditions comprise trauma, rheumatoid arthritis and osteoarthritis. *
38. A process according to any one of claims 28-34, which is a process for detecting the formation of cartilage during embryonal development.
39. A process according to any one of claims 28-34, which is a process for detecting physiological or therapeutic reparation of cartilage.
40. A process according to any one of claims 28-34, which is a process for selection and analysis, or for sorting, isolating or purification of chondrocytes.
41. A process according to any one of claims 28-34, which is a process for detecting regeneration of cartilage or chondrocytes during transplantation of cartilage or chondrocytes.
42. A process according to any one of claims 28-34, which is a process for in vitro studies of differentiation of chondrocytes.
43. A process of using binding entities having the capability of binding specifically to an integrin subunit ╬▒lO comprising the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, or an integrin heterodimer comprising said subunit ╬▒lO and a subunit ╬▓, or to homo- 56 logues or fragments thereof having similar biological activity, as markers or target molecules of cells or tissues expressing said integrin subunit ╬▒lO, which cells or tissues are of animal including human origin.
44. A process according to claim 43, whereby said fragment is a peptide chosen from the group comprising peptides of the cytoplasmic domain, the I-domain and the spliced domain.
45. A process according to claim 43, whereby said fragment is a peptide comprising the amino acid sequence
KLGFFAHKKIPEEEKREEKLEQ.
46. A process according to claim 43, whereby said fragment comprises the amino acid sequence from about amino acid no. 952 to about amino acid no. 986 of SEQ ID No. 1.
47. A process according to claim 43,' whereby said fragment comprises the amino acid sequence from about amino acid no. 140 to about amino acid No. 337 of
SEQ ID No. 1.
48. A process according to claim 43, whereby the subunit ╬▓ is ╬▓l .
49. A process according to any one of claims 43-48, which is a process for detecting the presence of an integrin subunit ╬▒lO comprising the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, or of an integrin heterodimer comprising said subunit ╬▒lO and a subunit ╬▓, or of homologues or fragments thereof having similar biological activity.
50. A process according to any one of claims 43-48, which process is a process for determining the differentiation-state of cells during embryonic development, angiogenesis, or development of cancer.
51. A process for detecting the presence of an inte¬ grin subunit αlO, or of a homologue or fragment of said integrin subunit having similar biological activity, on cells, whereby a polynucleotide or oligonucleotide chosen from the group comprising a polynucleotide or oligo- 57 nucleotide shown in SEQ ID No. 1 is used as a marker under hybridisation conditions wherein said polynucleotide or oligonucleotide fails to hybridise to a DNA or RNA encoding an integrin subunit αl .
52. A process according to claim 51, whereby said cells are chosen from the group comprising chondrocytes, smooth muscle cells, endothelial cells, osteoblasts and fibroblasts .
53. A process according to claim 51, whereby said fragment is a peptide chosen from the group comprising peptides of the cytoplasmic domain, the I-domain and the spliced domain.
54. A process according to claim 53, whereby said fragment is a peptide comprising the amino acid sequence KLGFFAHKKIPEEEKREEKLEQ.
55. A process according to claim 53," whereby said fragment comprises the amino acid sequence from about amino acid No. 952 to about amino acid no. 986 of
SEQ ID No. 1.
56. A process according to claim 53, whereby said fragment comprises the amino acid sequence from about amino acid No. 140 to about amino acid No. 337 of SEQ ID No. 1.
57. A process according to any one of claims 43-48, which is a process for determining the differentiation- state of cells during development, in pathological conditions, in tissue regeneration or in therapeutic and physiological reparation of cartilage.
58. A process according to claim 57, wherein the pathological conditions are any pathological conditions involving the integrin subunit ╬▒lO.
59. A process according to claim 58, whereby said pathological conditions are rheumatoid arthritis, osteoarthrosis or cancer.
60. A process according to claim 57, whereby said cells are chosen from the group comprising chondrocytes, 58 smooth muscle cells, endothelial cells, osteoblasts and fibroblasts .
61. A process for determining the differentiation- state of cells during development, in pathological con- ditions, in tissue regeneration and in therapeutic and physiological reparation of cartilage, whereby a polynucleotide or oligonucleotide chosen from the nucleotide sequence shown in SEQ ID No. 1 is used as a marker under hybridisation conditions wherein said polynucleotide or oligonucleotide fails to hybridise to a DNA or RNA encoding an integrin subunit ╬▒l .
62. A process according to claim 61, whereby said polynucleotide or oligonucleotide is a polynucleotide or oligonucleotide coding for a peptide chosen from the group comprising peptides of the cytoplasmic domain, the I-domain and the spliced domain. *
63. A process according to claim 62, whereby said polynucleotide or oligonucleotide is a polynucleotide or oligonucleotide coding for a peptide comprising the amino acid sequence KLGFFAHKKIPEEEKREEKLEQ.
64. A process according to claim 62, whereby said peptide comprises the amino acid sequence from about amino acid no. 952 to about amino acid no. 986 of SEQ ID No. 1.
65. A process according to claim 62, whereby said peptide comprises the amino acid sequence from about amino acid no. 140 to about amino acid no. 337 of SEQ ID No. 1.
66. A process according to claim 61, whereby said pathological conditions are any pathological conditions involving the integrin subunit ╬▒lO.
67. A process according to claim 66, whereby said pathological conditions are rheumatoid arthritis, osteoarthrosis or cancer.
68. A process according to claim 66, whereby said pathological conditions are atherosclerosis or inflammation. 59
69. A process according to any one of claims 61-68, whereby said cells are chosen from the group comprising chondrocytes, smooth muscle cells, endothelial cells, osteoblasts and fibroblasts.
70. A pharmaceutical composition comprising as an active ingredient a pharmaceutical agent or an antibody which is capable of using an integrin heterodimer comprising a subunit ╬▒lO and a subunit ╬▓, or the subunit ╬▒lO thereof, or a homologue or fragment of said integrin or subunit ╬▒lO having similar biological activity, as a target molecule.
71. A pharmaceutical composition according to claim 70, for use in stimulating, inhibiting or blocking the formation of cartilage, bone or blood vessels.
72. A pharmaceutical composition according to claim 70, for use in preventing adhesion betweeh tendon/ligaments and the surrounding tissue after infection, inflammation and after surgical intervention where adhesion impairs the function of the tissue.
73. A vaccine comprising as an active ingredient an integrin heterodimer comprising a subunit ╬▒lO and a subunit ╬▓, or the subunit ╬▒lO thereof, or a homologue or fragment of said integrin or subunit ╬▒lO, or DNA or RNA coding for said integrin subunit ╬▒lO.
74. Use of the integrin subunit ╬▒lO as a marker or target in transplantation of cartilage or chondrocytes.
75. A method of using binding entities having the capability of binding specifically to an integrin subunit ╬▒lO comprising the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, or an integrin heterodimer comprising said subunit ╬▒lO and a subunit ╬▓, or to homologues or fragments thereof having similar biological activity, for promoting adhesion of chondrocytes and/or osteoblasts to surfaces of implants to stimulate osseo- integration.
76. Use of an integrin heterodimer comprising an integrin subunit ╬▒lO and a subunit ╬▓, or the subunit ╬▒lO 60 thereof, or a homologue or fragment of said integrin or subunit ╬▒lO having similar biological activity, as a target for anti-adhesive drugs or molecules in tendon, ligament, skeletal muscle or other tissues where adhesion impairs the function of the tissue.
77. A method of stimulating, inhibiting or blocking the formation of cartilage or bone, comprising administration to a subject a suitable amount of a pharmaceutical agent or an antibody which is capable of using an integrin heterodimer comprising a subunit ╬▒lO and a subunit ╬▓, or the subunit ╬▒lO thereof, or a homologue or fragment of said integrin or subunit ╬▒lO having similar biological activity, as a target molecule.
78. A method of preventing adhesion between tendon/ ligaments and the surrounding tissue after infection, inflammation and after surgical intervention where adhesion impairs the function of the tissue, comprising administration to a subject a suitable amount of a pharmaceutical agent or an antibody which is capable of using a integrin heterodimer comprising a subunit ╬▒lO and a subunit ╬▓, or the subunit ╬▒lO thereof, or a homologue or fragment of said integrin or subunit ╬▒lO having similar biological activity, as a target molecule.
79. A method of stimulating extracellular matrix synthesis and repair by activation or blockage of an integrin heterodimer comprising a subunit ╬▒lO and a subunit ╬▓, or of the subunit ╬▒lO thereof, or of a homologue or fragment of said integrin or subunit ╬▒lO having similar biological activity.
80. A method of in vitro detecting the presence of integrin binding entities, comprising interaction of an integrin heterodimer comprising a subunit ╬▒lO and a subunit ╬▓, or the subunit ╬▒lO thereof, or a homologue or fragment of said integrin or subunit, with a sample, thereby causing said integrin, subunit ╬▒lO, or homologue or fragment thereof having similar biological activity, 61 to modulate the binding to its natural ligand or other integrin binding proteins present in said sample.
81. A method of in vitro studying consequences of the interaction of a human heterodimer integrin compris- ing a subunit ╬▒lO and a subunit ╬▓, or the subunit ╬▒lO thereof, or a homologue or fragment of said integrin or subunit, with an integrin binding entity and thereby initiate a cellular reaction.
82. A method according to claim 81, whereby the con- sequences of said interactions are measured as alterations in cellular functions.
83. A method of using DNA or RNA encoding an integrin subunit ╬▒lO or homologues or fragments thereof as a target molecule.
84. A method according to claim 83, whereby a polynucleotide or oligonucleotide hybridises to the DNA or RNA encoding an integrin subunit ╬▒lO or homologues or fragments thereof and whereby said polynucleotide or oligonucleotide fails to hybridise to a DNA or RNA encoding en integrin subunit ╬▒l.
85. A method of using a human heterodimer integrin comprising a subunit ╬▒lO and a subunit ╬▓, or the subunit ╬▒lO thereof, or a homologue or fragment of said integrin or subunit, or a DNA or RNA encoding an integrin subunit ╬▒lO or homologues or fragments thereof, as a marker or target molecule during angiogenesis.
86. A pharmaceutical composition comprising as an active ingredient a pharmaceutical agent or an antibody which is capable of stimulating cell surface expression of an integrin heterodimer comprising a subunit ╬▒lO and a subunit ╬▓, or the subunit ╬▒lO thereof, or a homologue or fragment of said integrin or subunit ╬▒lO having similar biological activity.
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US09/647,544 US7029858B1 (en) 1998-04-02 1999-03-31 Integrin heterodimer and a subunit thereof
EP99919725A EP1068238B1 (en) 1998-04-02 1999-03-31 An integrin heterodimer and a subunit thereof
JP2000542360A JP4912528B2 (en) 1998-04-02 1999-03-31 Integrin heterodimer and its subunits
CA2324381A CA2324381C (en) 1998-04-02 1999-03-31 An integrin heterodimer and a subunit thereof
ES99919725T ES2396693T3 (en) 1998-04-02 1999-03-31 Heterodimer of integrin and subunit thereof
AU37380/99A AU761430B2 (en) 1998-04-02 1999-03-31 An integrin heterodimer and a subunit thereof
US11/347,179 US8048991B2 (en) 1998-04-02 2006-02-06 Antibody to an integrin heterodimer and/or a subunit thereof
US13/226,323 US20120136141A1 (en) 1998-04-02 2011-09-06 Integrin Heterodimer And A Subunit Thereof
US13/651,602 US8895253B2 (en) 1998-04-02 2012-10-15 Integrin heterodimer and a subunit thereof
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